Codon-optimized polynucleotide-based vaccines against human cytomegalovirus infection

ABSTRACT

The invention is related to polynucleotide-based cytomegalovirus vaccines. In particular, the invention is plasmids operably encoding HCMV antigens, in which the naturally-occurring coding regions for the HCMV antigens have been modified for improved translation in human or other mammalian cells through codon optimization. HCMV antigens which are useful in the invention include, but are not limited to pp65, glycoprotein B (gB), IE1, and fragments, variants or derivatives of either of these antigens. In certain embodiments, sequences have been deleted, e.g., the Arg435-Lys438 putative kinase in pp65 and the membrane anchor and endocellular domains in gB. The invention is further directed to methods to induce an immune response to HCMV in a mammal, for example, a human, comprising delivering a plasmid encoding a codon-optimized HCMV antigen as described above. The invention is also directed to pharmaceutical compositions comprising plasmids encoding a codon-optimized HCMV antigen as described above, and further comprising adjuvants, excipients, or immune modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 13/525,814, filed Jun. 18, 2012, which is acontinuation application of U.S. application Ser. No. 13/013,752, filedJan. 25, 2011, now U.S. Pat. No. 8,278,093, issued Oct. 2, 2012, whichis a continuation application of U.S. application Ser. No. 11/892,020,filed Aug. 17, 2007, now U.S. Pat. No. 7,888,112, issued Feb. 15, 2011,which is a continuation application of U.S. application Ser. No.10/738,986, filed Dec. 19, 2003, now U.S. Pat. No. 7,410,795, issuedAug. 12, 2008, which claims benefit of U.S. Provisional Application No.60/435,549, filed Dec. 23, 2002, all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Human cytomegalovirus (“HCMV”) infects between 50% and 85% of adults by40 years of age (Gerson A. A., et al., in Viral Infections of Humans,Evans A. S. and Kaslow, R. A., eds., Plenum Press, New York, N.Y.(1997)). Although HCMV infection is benign in most healthy adults, itcan result in deadly pneumonitis, as well as colitis, esophagitis,leukopenia, and retinitis in transplant and other immuno-compromisedpatients, especially those with HIV. In solid organ transplant (SOT) orhematopoeitic cell transplant (HCT) populations, HCMV disease can occureither from new infection transmitted from the donor organ or HCT, orcan recur as a result of reactivation of latent virus in the recipient.

Despite licensed therapies, HCMV-associated disease remains severelydebilitating and life-threatening in HIV patients and the allogeneicrelated HCT and SOT settings. In addition, HCMV is the most commonintrauterine infection in the United States, and results in death orsevere sequelae in over 8,000 infants per year. For these reasons, HCMVwas ranked in the list of the top 10 vaccines most in need ofdevelopment in the United States (Vaccines for the 21^(st) century: atool for decision making, National Academy of Sciences (1999)).

Existing therapies include the use of immunoglobulins and anti-viralagents such as ganciclovir and its derivatives, which are most effectivewhen used prophylactically or very early during infection in at riskpopulations. However, these therapies are characterized by significanttoxicity and limited efficacy, especially for late onset disease (onsetafter the first 100 days) (Fillet, A. M., Drugs Aging 19:343-354 (2002);von Bueltzingsloewen, A., et al., Bone Marrow Transplant 12:197-202(1993); Winston, D. J., et al., Ann. Intern. Med. 118:179-184 (1993);Goodrich, J. M., et al., Ann. Intern. Med. 118:173-178 (1993); Boeckh,M., et al., Blood 88:4063-4071 (1996); Salzberger, B., et al., Blood90:2502-2508 (1997); Preiser, W., et al., J. Clin. Virol. 20:59-70(2001); Grangeot-Keros, L., and Cointe, D., J. Clin. Virol. 21:213-221(2001); Boeckh, M., and Bowden, R., Cancer Treat. Res. 76:97-136 (1995);Zaia, J. A., et al., Hematology (Am. Soc. Hematol. Educ. Program)339-355 (2000)).

In addition to developing more rapid and sensitive diagnostics,molecular biological methods enable the development of defined subunitvaccines for human pathogens. Indeed, safe, effective recombinantsubunit vaccines would significantly reduce, and perhaps eliminate, theneed for therapeutic treatments. In the case of HCMV, control ofinfection has been correlated with antibody and T cell recognition of atleast three viral proteins: pp65, glycoprotein B (gB), and the immediateearly-1 protein (IE1).

The 65kD viral protein pp65, also known as ppUL83, lower matrix protein,ICP27, PK68, and pp64, is one of the most abundantly expressedstructural proteins (FIG. 1). It is encoded by the UL83 gene of theviral genome (nucleotides 119352-121037 of the HCMV strain AD169 genomicsequence, Genbank X17403). This protein is believed to be processed forMHC presentation shortly after viral entry into cells, which allows itto be presented before other viral proteins shut down the antigenprocessing pathway in infected cells. Therefore, T cell recognition ofthis protein is important for infection control (Solache, et al. J.Immunol. 163:5512-5518 (1999)), which is herein incorporated byreference in its entirety.

Glycoprotein B (gB) is a 906 amino acid envelope glycoprotein (FIG. 4)encoded by UL55, nucleotides 80772-83495 of Genbank X17403). It is atype I integral membrane protein that participates in the fusion of thevirion envelope with the cell membrane, is required for infectivity, ishighly immunogenic, and has a high degree of conservation among HCMVstrains, making this protein an attractive target for vaccines. Thefull-length protein contains an amino-terminal signal peptide (aminoacids 1-24), an extracellular domain (amino acids 25-713), a putativetrans-membrane anchor domain (amino acids 714-771) and an intracellulardomain (amino acids 772-906). Deletion of the transmembrane anchordomain results in secretion of gB (Zheng et al. J. Virol. 70:8029-8040(1996)). Additionally, the full-length protein is cleaved by host furinproteases between amino acids 460 and 461 to form the gp93 and gp55cleavage products that remain tightly associated as a heterodimer.(Mocarski E. S. and C. T. Courcelle, pp. 2629-2674, Field's Virology,4th ed., Eds. Knipe D M and Howley P M, Lippincott Williams & Wilkins,Philadelphia (2001)). Each of the references cited in this paragraph isincorporated herein by reference in its entirety.

IE1 is a 491 amino acid protein (FIG. 7) encoded by HCMV ORF UL123(Genbank X17403, nucleotides 171006-172765). The gene encodes a 1.9 KbmRNA comprising four exons, with only exons 2-4 being translated. The 85N-terminal amino acids are encoded by exons 2 and 3, with the remainderencoded by exon 4. IE2 is a related family of proteins that share exons1-3 and an exon 5, with many splice variations. Together, IE1 and IE2transactivate the HCMV major immediate early promoter to regulate viraltranscription (Malone, C L. et al. J. Virol. 64:1498-1506 (1990);Mocarski, E. Fields Virology Ed. Field et al., 3^(rd) ed., pp.2447-2491, Lippincott-Raven Publishers, Philadelphia (1996); Chee M. S.et al., Curr Topics Microbiol. Immunol. 154:125-169 (1990)). Each of thereferences cited in this paragraph is incorporated herein by referencein its entirety.

IE1 has a kinase activity that is dependent on an ATP binding siteencoded by amino acids 173-196. IE1 can autophosphorylate orphosphorylate cellular factors to transactivate E2F dependenttranscription. Both exons 3 and 4 are required for viraltransactivation, with the required regions in exon 4 being broadlydistributed throughout the exon. The portion of the protein encoded byexon 4 is known to have a high degree of secondary structure. AlthoughIE1 is transported to the nucleus, no nuclear localization signal hasbeen identified. (Pajovic, S. et al. Mol. Cell. Bio. 17:6459-6464(1997)). Gyulai et al. showed high levels of CTL response in vitro toeffector cells expressing a nucleotide fragment consisting of exon 4(Gyulai et al. J. Infectious Diseases 181:1537-1546 (2000)). Each of thereferences cited in this paragraph is incorporated herein by referencein its entirety.

No vaccine is currently available for HCMV. However, clinical trialshave been performed with live-attenuated HCMV vaccines, acanarypox-based vaccine, and a recombinant gB vaccine (Plotkin, S. A.,Pediatr. Infect. Dis. J. 18:313-325 (1999)). The first HCMV vaccinetested in humans was a live attenuated virus vaccine made from the AD169laboratory-adapted strain (Elek, S. D. and Stern, H., Lancet 1:1-5(1974)). Local reactions were common, but HCMV was not isolated from anyof the vaccine recipients. This vaccine was not investigated beyondinitial Phase I studies.

Immune responses to HCMV have been determined by the study of acute andchronic HCMV infections in both animal models and in man. Antibodyappears critical in the prevention of maternal-fetal transmission, andis primarily directed to the envelope glycoproteins, especially gB(Plotkin, S. A., Pediatr. Infect. Dis. J. 18:313-325 (1999); Fowler, K.B., N. Engl. J. Med. 326:663-667 (1992)).

In contrast, the control of HCMV infection in transplant recipients andHIV-infected persons is associated with preserved cellular immuneresponses, including CD4+, CD8+, and NK T cells. The CD8+ T-cellresponses are directed primarily at the immediate early (IE) protein ofHCMV and at the abundant tegument protein pp65 (Gyulai, Z., et al., J.Infect. Dis. 181:1537-1546 (2000); Tabi, Z., et al., J. Immunol.166:5695-5703 (2001); Wills, M. R., et al., J. Virol. 70:7569-7579(1996); Frankenberg, N., et al., Virology 295:208-216 (2002); Retiere,C., et al., J. Virol. 74:3948-3952 (2000); Koszinowski, U. H., et al.,J. Virol. 61:2054-2058 (1987); Kern, F., et al., J. Infect. Dis.185:1709-1716 (2002)). Approximately 92% of persons have CD8+ responsesto pp65 and another 76% to exon 4 of IE1 (Gyulai, Z., et al., J. Infect.Dis. 181:1537-1546 (2000); Kern, F., et al., J. Infect. Dis.185:1709-1716 (2002)). In addition, another one third of infectedindividuals have CTL responses to gB. Almost all infected persons haveCD4+ responses to HCMV, although the gene and epitope mapping of theseresponses is not as fully investigated as those for CD8+ T cells (Kern,F., et al., J. Infect. Dis. 185:1709-1716 (2002); Davignon, J. L., etal., J. Virol. 70:2162-2169 (1996); He, H., et al., J. Gen. Virol.76:1603-1610 (1995); Beninga, J., et al., J. Gen. Virol. 76:153-160(1995). The helper T-cell responses in infected, healthy persons aresufficiently robust that HCMV is frequently used as a positive controlin the development of methods for the measurement of CD4+ T-cellresponses (Kern, F., et al., J. Infect. Dis. 185:1709-1716 (2002);Currier, J. R., et al., J. Immunol. Methods 260:157-172 (2002); Picker,L. J., et al., Blood 86:1408-1419 (1995)).

Other attempts to develop vaccines for HCMV have focused onadministering purified or recombinant viral polypeptides, eitherfull-length, modified, or short epitopes, to induce immune responses. Ina review published by the American Society for Hematology, Zaia et al.describes various peptide-based approaches to developing HCMV vaccines,including using DNA vaccines to express wild-type and mutated proteins(Zaia, J. A. et al. Hematology 2000, Am Soc Hematol Educ Program, pp.339-355, Am. Soc. Hematol. (2000)). Endresz et al. describes elicitingHCMV-specific CTL in mice immunized with plasmids encoding HCMV Townestrain full-length gB, expressed constitutively or under atetracycline-regulatable promoter, and pp65 or a gB with the deletion ofamino acids 715-772 (Endresv, V. et al. Vaccine 17:50-8 (1999); Endresz,V. et al. Vaccine 19:3972-80 (2001)). U.S. Pat. No. 6,100,064 describesa method of producing secreted gB polypeptides lacking the transmembranedomain but retaining the C terminal domain. U.S. Pat. Nos. 5,547,834 and5,834,307 describe a gB polypeptide with amino acid substitutions at theendoproteolytic cleavage site to prevent proteolytic processing. U.S.Pat. Nos. 6,251,399 and 6,156,317 describe vaccines using short peptidefragments of pp65 comprising immunogenic epitopes. A number of othergroups have analyzed epitopes in HCMV pp65 and gB for eliciting a strongimmune response (Liu, Y N. et al. J. Gen. Virol. 74:2207-14 (1993);Ohlin, M. et al. J. Virol. 67:703-10 (1993); Navarro, D. et al. J. Med.Virol. 52:451-9 (1997); Khattab B A. et al. J. Med. Virol. 52:68-76(1997); Diamond, D J. et al. Blood 90:1751067 (1997); Solache, A. et al.J. Immunol. 163:5512-8 (1999). U.S. Pat. No. 6,162,620 is directed to apolynucleotide encoding a wild-type gB or a gB lacking the membranesequences. U.S. Pat. No. 6,133,433 is directed to a nucleotide encodinga full-length, wild-type pp65 or a specific 721 nt fragment thereof.Each of the references cited in this paragraph is incorporated herein byreference in its entirety.

During the past few years there has been substantial interest in testingDNA-based vaccines for a number of infectious diseases where the needfor a vaccine, or an improved vaccine, exists. Several well-recognizedadvantages of DNA-based vaccines include the speed, ease and cost ofmanufacture, the versatility of developing and testing multivalentvaccines, the finding that DNA vaccines can produce a robust cellularresponse in a wide variety of animal models as well as in man, and theproven safety of using plasmid DNA as a delivery vector (Donnelly, J.J., et al., Annu. Rev. Immunol. 15:617-648 (1997); Manickan, E., et al.,Crit. Rev. Immunol. 17(2):139-154 (1997)). DNA vaccines represent thenext generation in the development of vaccines (Nossal, G., Nat. Med.4:475-476 (1998)) and numerous DNA vaccines are in clinical trials. Eachof the references cited in this paragraph is incorporated herein byreference in its entirety.

The immunotherapeutic product design is based on the concept ofimmunization by direct gene transfer. Plasmid-based immunotherapeuticsoffer the positive attributes of immune stimulation inherent tolive-attenuated vaccines combined with the safety of recombinant subunitvaccines in an adjuvant formulation.

In the transplant population, control of HCMV disease is associated witha cellular immune response (Riddell, S. R., “Pathogenesis ofcytomegalovirus pneumonia in immunocompromised hosts,” Semin. Respir.Infect. 10:199-208 (1995)) and thus an effective product should induceCD4+ and CD8+ T-cell responses. Formulated plasmid has been shown toinduce such cellular immune responses, and does not have the safetyconcerns associated with the use of live vectors in the transplantsetting (Shiver, J. W., et al., Nature 415:331-335 (2002)).

Retooling coding regions encoding polypeptides from pathogens usingcodon frequencies preferred in a given mammalian species often resultsin a significant increase in expression in the cells of that mammalianspecies, and concomitant increase in immunogenicity. See, e.g., Deml,L., et al., J. Virol. 75:10991-11001 (2001), and Narum, D L, et al.,Infect. Immun. 69:7250-7253 (2001), all of which are herein incorporatedby reference in its entirety.

There remains a need in the art for convenient, safe, and efficaciousimmunogenic compounds to protect humans against HCMV infection. Thepresent invention provides safe yet effective immunogenic compounds andmethods to protect humans, especially transplant recipients andimmunocompromised individuals, against HCMV infection using suchimmunogenic compounds.

SUMMARY OF THE INVENTION

The present invention is directed to enhancing immune response of ahuman in need of protection against HCMV infection by administering invivo, into a tissue of the human, a polynucleotide comprising acodon-optimized coding region encoding an HCMV polypeptide or a nucleicacid fragment of such a coding region encoding a fragment, a variant, ora derivative thereof. Nucleic acid fragments of the present inventionare altered from their native state in one or more of the followingways. First, a nucleic acid fragment which encodes an HCMV polypeptidemay be part or all of a codon-optimized coding region, optimizedaccording to codon usage in humans. In addition, a nucleic acid fragmentwhich encodes an HCMV polypeptide may be a fragment which encodes only aportion of a full-length polypeptide, and/or may be mutated so as to,for example, remove from the encoded polypeptide adventitious proteinmotifs present in the encoded polypeptide or virulence factorsassociated with the encoded polypeptide. For example, the nucleic acidsequence could be mutated so as not to encode adventitious anchoringmotifs that prevent secretion of the polypeptide. Upon delivery, thepolynucleotide of the invention is incorporated into the cells of thehuman in vivo, and a prophylactically or therapeutically effectiveamount of an HCMV polypeptide or fragment thereof is produced in vivo.

The invention further provides immunogenic compositions comprising apolynucleotide which comprises one or more codon-optimized codingregions encoding polypeptides of HCMV or nucleic acid fragments of suchcoding regions encoding fragments, variants, or derivatives thereof.Such compositions may include various transfection facilitating orimmunity enhancing agents, such as poloxamers, cationic lipids, oradjuvants.

The present invention further provides plasmids and other polynucleotideconstructs for delivery of nucleic acid coding sequences to a vertebratewhich provide expression of HCMV polypeptides, or fragments, variants,or derivatives thereof. The present inventions further providescarriers, excipients, transfection-facilitating agents,immunogenicity-enhancing agents, e.g. adjuvants, or other agent oragents to enhance the transfection, expression, or efficacy of theadministered gene and its gene product.

The invention further provides methods for enhancing the immune responseof a human to HCMV infection by administering to the tissues of a humanone or more polynucleotides comprising one or more codon-optimizedcoding regions encoding polypeptides of HCMV or nucleic acid fragmentsof such coding regions encoding fragments, variants, or derivativesthereof. In certain embodiments, the invention further provides methodsfor enhancing the immune response of a human patient to HCMV infectionby sequentially administering two or more different immunogeniccompositions to the tissues of the vertebrate. Such methods compriseinitially administering one or more polynucleotides comprising one ormore codon-optimized coding regions encoding polypeptides of HCMV ornucleic acid fragments of such coding regions encoding fragments,variants, or derivatives thereof, to prime immunity, and thenadministering subsequently a different vaccine composition, for examplea recombinant viral vaccine, a protein subunit vaccine, or a recombinantor killed bacterial vaccine or vaccines to boost the anti-HCMV immuneresponse in a human.

The invention further provides methods for enhancing the immune responseof a human patient to HCMV by administering to the tissues of a humanone or more polynucleotides comprising one or more codon-optimizedcoding regions encoding polypeptides of HCMV, and also HCMV polypeptidesor fragments, variants or derivatives thereof; or one or morenon-optimized polynucleotides encoding HCMV polypeptides, fragments,variants or derivatives thereof.

The combination of HCMV polypeptides or polynucleotides encoding HCMVpolypeptides or fragments, variants or derivatives thereof, with thecodon-optimized nucleic acid compositions provides for therapeuticallybeneficial effects at dose sparing concentrations. For example,immunological responses sufficient for a therapeutically beneficialeffect may be attained by using less of a conventional-type vaccine whensupplemented or enhanced with the appropriate amount of acodon-optimized nucleic acid.

Conventional-type vaccines, include vaccine compositions comprisingeither dead, inert or fragments of a virus or bacteria, or bacterial orviral proteins or protein fragments, injected into the patient to elicitaction by the immune system. With regard to the present invention,conventional-type vaccines include compositions comprising immunogenicpolypeptides or nucleotides encoding immunogenic polypeptides,fragments, variants, or derivatives thereof, and vectors comprisingnucleotides encoding immunogenic polypeptides, fragments, variants, orderivatives thereof, that are not products of, or do not containcodon-optimized polynucleotides as described herein. Thus, geneticallyengineered vaccines, are included in conventional-type vaccines, such asgenetically engineered live vaccines, live chimeric vaccines, livereplication-defective vaccines, subunit vaccines, peptide vaccines invarious modifications of monovalent, multivalent, or chimeric subunitvaccines delivered as individual components or incorporated intovirus-like particles for improved immunogenicity, and polynucleotidevaccines. Auxiliary agents, as described herein, are also consideredcomponents of conventional-type vaccines.

Thus, dose sparing is contemplated by administration of thecombinatorial polynucleotide vaccine compositions of the presentinvention.

In particular, the dose of conventional-type vaccine may be reduced byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered incombination, or prior to, or subsequent to, the codon-optimized nucleicacid compositions of the invention.

Similarly, a desirable level of an immunological response afforded by aDNA based pharmaceutical alone may be attained with less DNA byincluding a conventional-type DNA vaccine. Further, using a combinationof a conventional-type vaccine and a codon-optimized DNA-based vaccinemay allow both materials to be used in lesser amounts while stillaffording the desired level of immune response arising fromadministration of either component alone in higher amounts (e.g. one mayuse less of either immunological product when they are used incombination). This reduction in amounts of materials being delivered maybe for each administration, in addition to reducing the number ofadministrations, in a vaccination regimen (e.g. 2 versus 3 or 4injections). Further, the combination may also provide for reducing thekinetics of the immunological response (e.g. desired response levels areattained in 3 weeks instead of 6 after immunization).

In particular, the dose of a DNA based pharmaceutical, may be reduced byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered in combinationwith conventional CMV vaccines.

Determining the precise amounts of DNA based pharmaceutical andconventional antigen is based on a number of factors as describedherein, and is readily determined by one of ordinary skill in the art.

In addition to dose sparing, the claimed combinatorial compositionsprovide for a broadening of the immune response and/or enhancedbeneficial immune responses. Such broadened or enhanced immune responsesare achieved by: adding DNA to enhance cellular responses to aconventional-type vaccine; adding a conventional-type vaccine to a DNApharmaceutical to enhanced humoral response; using a combination thatinduces additional epitopes (both humoral and/or cellular) to berecognized and/or more desirably responded to (epitope broadening);employing a DNA-conventional vaccine combination designed for aparticular desired spectrum of immunological responses; obtaining adesirable spectrum by using higher amounts of either component. Thebroadened immune response is measurable by one of ordinary skill in theart by various standard immunological assays specific for the desirableresponse spectrum, which are described in more detail herein.

Both broadening and dose sparing may be obtained simultaneously.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the wild-type nucleotide sequence (SEQ ID NO:1) and aminoacid translation (SEQ ID NO:2) of full-length, native HCMV pp65 (GenbankWMBE65) from HCMV strain AD 169.

FIG. 2 shows a fully codon-optimized nucleotide sequence (SEQ ID NO:3)and amino acid translation (SEQ ID NO:4) of native HCMV pp65. Theputative kinase site at amino acids Arg435-Lys438 is underlined.

FIGS. 3A-3E show the alignment of wild-type (“wt”) (SEQ ID NO:1) andfully codon-optimized (“opt”) (SEQ ID NO:9) nucleotide sequencesencoding native HCMV pp65.

FIGS. 4A-4E show the wild-type nucleotide sequence (SEQ ID NO:11) andamino acid translation (SEQ ID NO:12) of HCMV gB strain AD 169. SEQ IDNO:11 contains a nucleic acid fragment encoding the open reading framefor full-length HCMV gB (nucleotides 157-3125 of Genbank X04606). Thehost proteolytic cleavage site between amino acids 460 and 461 is markedby a colon.

FIGS. 5A-5D show a minimally codon-optimized nucleotide sequence (SEQ IDNO:13) and amino acid sequence (SEQ ID NO:14) of a truncated, secretedHCMV gB. SEQ ID NO:13 contains a nucleic acid encoding a minimal humancodon-optimized secreted gB (SEQ ID NO:14).

FIGS. 6A-6G show the alignment of wild-type (“wt”) (SEQ ID NO:11) andfully codon-optimized (“opt”) (SEQ ID NO:16) nucleotide sequencesencoding full-length wild-type HCMV gB.

FIGS. 7A-7B show the wild-type IE1 nucleotide sequence (SEQ ID NO:19),and amino acid translation (SEQ ID NO:20) of full-length, native IE1.

FIG. 8 shows the protocol for the preparation of a formulationcomprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a finalvolume of 3.6 ml, through the use of thermal cycling.

FIG. 9 shows the protocol for the preparation of a formulationcomprising 0.3 mM BAK, 34 mg/ml or 50 mg/ml CRL 1005, and 2.5 mg/ml DNAin a final volume of 4.0 ml, through the use of thermal cycling.

FIG. 10 shows the protocol for the simplified preparation (withoutthermal cycling) of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL1005, and 5 mg/ml DNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods forenhancing the immune response of a human in need of protection againstHCMV infection by administering in vivo, into a tissue of a human, apolynucleotide comprising a human codon-optimized coding region encodinga polypeptide of HCMV, or a nucleic acid fragment of such a codingregion encoding a fragment, variant, or derivative thereof. Thepolynucleotides are incorporated into the cells of the human in vivo,and an immunologically effective amount of the HCMV polypeptide, orfragment or variant is produced in vivo.

The present invention provides polynucleotide-based vaccines and methodsfor delivery of HCMV coding sequences to a human with optimal expressionand safety conferred through codon optimization and/or othermanipulations. These polynucleotide-based vaccines are prepared andadministered in such a manner that the encoded gene products areoptimally expressed in humans. As a result, these compositions andmethods are useful in stimulating an immune response against HCMVinfection. Also included in the invention are expression systems,delivery systems, and codon-optimized HCMV coding regions.

A polynucleotide vaccine of the present invention is capable ofeliciting an immune response in a human against HCMV when administeredto that human. Such polynucleotides are referred to herein aspolynucleotide vaccines.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a polynucleotide,” is understood torepresent one or more polynucleotides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

The terms “nucleic acid” or “nucleic acid fragment” refers to any one ormore nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide or construct. While the terms “nucleic acid,” as usedherein, is meant to include any nucleic acid, the term “nucleic acidfragment” is used herein to specifically denote a fragment of a designedor synthetic codon-optimized coding region encoding a polypeptide, orfragment, variant, or derivative thereof, which has been optimizedaccording to the codon usage of a given species. As used herein, a“coding region” is a portion of nucleic acid which consists of codonstranslated into amino acids. Although a “stop codon” (TAG, TGA, or TAA)is not translated into an amino acid, it may be considered to be part ofa coding region, but any flanking sequences, for example promoters,ribosome binding sites, transcriptional terminators, and the like, arenot part of a coding region. Two or more nucleic′ acids or nucleic acidfragments of the present invention can be present in a singlepolynucleotide construct, e.g., on a single plasmid, or in separatepolynucleotide constructs, e.g., on separate plasmids. Furthermore, anynucleic acid or nucleic acid fragment may encode a single polypeptide,e.g., a single antigen, cytokine, or regulatory polypeptide, or mayencode more than one polypeptide, e.g., a nucleic acid may encode two ormore polypeptides. In addition, a nucleic acid may encode a regulatoryelement such as a promoter or a transcription terminator, or may encodeheterologous coding regions, e.g. specialized elements or motifs, suchas a secretory signal peptide or a functional domain.

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to HCMV polypeptides of the present invention include anypolypeptides which retain at least some of the immunogenicity orantigenicity of the corresponding native polypeptide. Fragments of HCMVpolypeptides of the present invention include proteolytic fragments,deletion fragments and in particular, fragments of HCMV polypeptideswhich exhibit increased secretion from the cell or higher immunogenicitywhen delivered to an animal. Polypeptide fragments further include anyportion of the polypeptide which comprises an antigenic or immunogenicepitope of the native polypeptide, including linear as well asthree-dimensional epitopes. Variants of HCMV polypeptides of the presentinvention includes fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally, such as anallelic variant. By an “allelic variant” is intended alternate forms ofa gene occupying a given locus on a chromosome or genome of an organismor virus. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985),which is incorporated herein by reference. For example, as used herein,variations in a given gene product, e.g., pp65, between HCMV strains,e.g. Towne and AD169, would be considered “allelic variants.”Non-naturally occurring variants may be produced using art-knownmutagenesis techniques. Variant polypeptides may comprise conservativeor non-conservative amino acid substitutions, deletions or additions.Derivatives of HCMV polypeptides of the present invention, arepolypeptides which have been altered so as to exhibit additionalfeatures not found on the native polypeptide. Examples include fusionproteins. An analog is another form of an HCMV polypeptide of thepresent invention. An example is a proprotein which can be activated bycleavage of the proprotein to produce an active mature polypeptide.

The term “polynucleotide” is intended to encompass a singular nucleicacid or nucleic acid fragment as well as plural nucleic acids or nucleicacid fragments, and refers to an isolated molecule or construct, e.g., avirus genome (e.g., a non-infectious viral genome), messenger RNA(mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles asdescribed in (Darquet, A-M et al., Gene Therapy 4:1341-1349 (1997))comprising a polynucleotide. A nucleic acid may be provided in linear(e.g., mRNA), circular (e.g., plasmid), or branched form as well asdouble-stranded or single-stranded forms. A polynucleotide may comprisea conventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)).

The terms “infectious polynucleotide” or “infectious nucleic acid” areintended to encompass isolated viral polynucleotides and/or nucleicacids which are solely sufficient to mediate the synthesis of completeinfectious virus particles upon uptake by permissive cells. “Isolated”means that the viral nucleic acid does not require pre-synthesizedcopies of any of the polypeptides it encodes, e.g., viral replicases, inorder to initiate its replication cycle.

The terms “non-infectious polynucleotide” or “non-infectious nucleicacid” as defined herein are polynucleotides or nucleic acids whichcannot, without additional added materials, e.g., polypeptides, mediatethe synthesis of complete infectious virus particles upon uptake bypermissive cells. An infectious polynucleotide or nucleic acid is notmade “non-infectious” simply because it is taken up by a non-permissivecell. For example, an infectious viral polynucleotide from a virus withlimited host range is infectious if it is capable of mediating thesynthesis of complete infectious virus particles when taken up by cellsderived from a permissive host (i.e., a host permissive for the virusitself). The fact that uptake by cells derived from a non-permissivehost does not result in the synthesis of complete infectious virusparticles does not make the nucleic acid “non-infectious.” In otherwords, the term is not qualified by the nature of the host cell, thetissue type, or the species.

In some cases, an isolated infectious polynucleotide or nucleic acid mayproduce fully-infectious virus particles in a host cell population whichlacks receptors for the virus particles, i.e., is non-permissive forvirus entry. Thus viruses produced will not infect surrounding cells.However, if the supernatant containing the virus particles istransferred to cells which are permissive for the virus, infection willtake place.

The terms “replicating polynucleotide” or “replicating nucleic acid” aremeant to encompass those polynucleotides and/or nucleic acids which,upon being taken up by a permissive host cell, are capable of producingmultiple, e.g., one or more copies of the same polynucleotide or nucleicacid. Infectious polynucleotides and nucleic acids are a subset ofreplicating polynucleotides and nucleic acids; the terms are notsynonymous. For example, a defective virus genome lacking the genes forvirus coat proteins may replicate, e.g., produce multiple copies ofitself, but is NOT infectious because it is incapable of mediating thesynthesis of complete infectious virus particles unless the coatproteins, or another nucleic acid encoding the coat proteins, areexogenously provided.

In certain embodiments, the polynucleotide, nucleic acid, or nucleicacid fragment is DNA. In the case of DNA, a polynucleotide comprising anucleic acid which encodes a polypeptide normally also comprises apromoter operably associated with the polypeptide-encoding nucleic acid.An operable association is when a nucleic acid encoding a gene product,e.g., a polypeptide, is associated with one or more regulatory sequencesin such a way as to place expression of the gene product under theinfluence or control of the regulatory sequence(s). Two DNA fragments(such as a polypeptide-encoding nucleic acid and a promoter associatedwith the 5′ end of the nucleic acid) are “operably associated” ifinduction of promoter function results in the transcription of mRNAencoding the desired gene product and if the nature of the linkagebetween the two DNA fragments does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product, or(3) interfere with the ability of the DNA template to be transcribed.Thus, a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), retroviruses (such as Rous sarcoma virus), andpicornaviruses (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). Other transcription controlregions include those derived from vertebrate genes such as actin, heatshock protein, bovine growth hormone and rabbit β-globin, as well asother sequences capable of controlling gene expression in eukaryoticcells. Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).

In one embodiment, a DNA polynucleotide of the present invention is acircular or linearized plasmid, or other linear DNA which is, in certainembodiments, non-infectious and nonintegrating (i.e., does not integrateinto the genome of vertebrate cells). A linearized plasmid is a plasmidthat was previously circular but has been linearized, for example, bydigestion with a restriction endonuclease.

Alternatively, DNA virus genomes may be used to administer DNApolynucleotides into vertebrate cells. In certain embodiments, a DNAvirus genome of the present invention is noninfectious, andnonintegrating. Suitable DNA virus genomes include herpesvirus genomes,adenovirus genomes, adeno-associated virus genomes, and poxvirusgenomes. References citing methods for the in vivo introduction ofnon-infectious virus genomes to vertebrate tissues are well known tothose of ordinary skill in the art, and are cited supra.

In other embodiments, a polynucleotide of the present invention is RNA.In a suitable embodiment, the RNA is in the form of messenger RNA(mRNA). Methods for introducing RNA sequences into vertebrate cells aredescribed in U.S. Pat. No. 5,580,859, the disclosure of which isincorporated herein by reference in its entirety.

Polynucleotide, nucleic acids, and nucleic acid fragments of the presentinvention may be associated with additional nucleic acids which encodesecretory or signal peptides, which direct the secretion of apolypeptide encoded by a nucleic acid or polynucleotide of the presentinvention. According to the signal hypothesis, proteins secreted bymammalian cells have a signal peptide or secretory leader sequence whichis cleaved from the mature protein once export of the growing proteinchain across the rough endoplasmic reticulum has been initiated. Thoseof ordinary skill in the art are aware that polypeptides secreted byvertebrate cells generally have a signal peptide fused to the N-terminusof the polypeptide, which is cleaved from the complete or “full-length”polypeptide to produce a secreted or “mature” form of the polypeptide.In certain embodiments, the native leader sequence is used, or afunctional derivative of that sequence that retains the ability todirect the secretion of the polypeptide that is operably associated withit. Alternatively, a heterologous mammalian leader sequence, or afunctional derivative thereof, may be used. For example, the wild-typeleader sequence may be substituted with the leader sequence of humantissue plasminogen activator (TPA) or mouse β-glucuronidase.

In accordance with one aspect of the present invention, there isprovided a plasmid for expression of an HCMV gB-derived or pp65-derivedcoding sequence optimized for expression in human cells, to be deliveredto a human to be treated or immunized. Additional HCMV-derived codingsequences, e.g. coding for IE1, may also be included on the plasmid, oron a separate plasmid, and expressed, either using native codons orcodons optimized for expression in humans to be treated or immunized.When such a plasmid encoding one or more optimized HCMV sequences isdelivered, in vivo to a tissue of the human to be treated or immunized,the transcriptional unit will thus express the one or more encoded geneproduct(s). The level of expression of the gene product(s) will dependto a significant extent on the strength of the associated promoter andthe presence and activation of an associated enhancer element, as wellas the optimization of the coding region.

As used herein, the term “plasmid” refers to a construct made up ofgenetic material (i.e., nucleic acids). Typically a plasmid contains anorigin of replication which is functional in bacterial host cells, e.g.,Eschericha coli, and selectable markers for detecting bacterial hostcells comprising the plasmid. Plasmids of the present invention mayinclude genetic elements as described herein arranged such that aninserted coding sequence can be transcribed and translated in eukaryoticcells. Also, while the plasmid may include a sequence from a viralnucleic acid, such viral sequence normally does not cause theincorporation of the plasmid into a viral particle, and the plasmid istherefore a non-viral vector. In certain embodiments described herein, aplasmid is a closed circular DNA molecule.

The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is translated to form a polypeptide product which has arelevant biological activity. Also, the process of expression mayinvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein,terms including, but not limited to “peptide,” “dipeptide,”“tripeptide,” “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are included inthe definition of a “polypeptide,” and the term “polypeptide” may beused instead of, or interchangeably with any of these terms. The termfurther includes polypeptides which have undergone post-translationalmodifications, for example, glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. Polypeptides, and fragments, derivatives, analogs,or variants thereof of the present invention can be antigenic andimmunogenic polypeptides related to HCMV polypeptides, which are used toprevent or treat, i.e., cure, ameliorate, lessen the severity of, orprevent or reduce contagion of infectious disease caused by HCMV.

As used herein, an antigenic polypeptide or an immunogenic polypeptideis a polypeptide which, when introduced into a human, reacts with thehuman's immune system molecules, i.e., is antigenic, and/or induces animmune response in the human, i.e., is immunogenic. It is quite likelythat an immunogenic polypeptide will also be antigenic, but an antigenicpolypeptide, because of its size or conformation, may not necessarily beimmunogenic. Examples of antigenic and immunogenic polypeptides of thepresent invention include, but are not limited to, HCMV pp65 orfragments or variants thereof, e.g. pp65-delArg435-Lys468; gB, orfragments thereof, e.g. consisting of amino acids 1-713, or variantsthereof; and IE1 or fragments or variants thereof, e.g. ex4-IE1-delATPand derivatives thereof, e.g., any of the foregoing polypeptides fusedto a TPA signal peptide.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, for example amammal, for example, a human. An “immunogenic epitope,” as used herein,is defined as a portion of a protein that elicits an immune response inan animal, as determined by any method known in the art. The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody can immunospecifically bind its antigen asdetermined by any method well known in the art. Immunospecific bindingexcludes non-specific binding but does not necessarily excludecross-reactivity with other antigens. Antigenic epitopes need notnecessarily be immunogenic.

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. Certain polypeptidescomprising immunogenic or antigenic epitopes are at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacid residues in length. Antigenic as well as immunogenic epitopes maybe linear, i.e., be comprised of contiguous amino acids in apolypeptide, or may be three dimensional, i.e., where an epitope iscomprised of non-contiguous amino acids which come together due to thesecondary or tertiary structure of the polypeptide, thereby forming anepitope.

As to the selection of peptides or polypeptides bearing an antigenicepitope (e.g., that contain a region of a protein molecule to which anantibody or T cell receptor can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, e.g., Sutcliffe, J. G., etal., Science 219:660-666 (1983).

Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longerpeptides, especially those containing proline residues, usually areeffective. Sutcliffe et al., supra, at 661. For instance, 18 of 20peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins. Non-limiting examples ofantigenic polypeptides or peptides for HCMV pp65, gB and IE1 epitopesknown to elicit cellular or humoral immune responses are listed in Table1.

TABLE 1 Epitopes for immune recognition for HCMV proteins pp65, gB, andIE1. All of the references herein are incorporated by reference in itsentirety. HCMV polypeptide Position Reference gB aa 178-194 Liu, YN. etal. J. Gen. Virol. 74: aa 190-204 2207-14 (1993) aa 250-264 aa 420-434gB aa 67-86 Ohlin, M. et al. J. Virol. 67: 703-10 aa 549-635 (1993). aa570-579 aa 606-619 gB aa 548-618 Navarro, D. et al. J. Med. Virol. 52:451-9 (1997) pp65 aa 361-376 Khattab BA. et al. J. Med. Virol. aa485-499 52: 68-76 (1997) pp65 aa 495-503 Diamond, DJ. et al. Blood 90:1751067 (1997) pp65 aa 14-22 Solache, A. et al. J. Immunol. aa 120-128163: 5512-8 aa 495-503 (1999) IE1 (UL123) aa 199-207 Khan, N. et al. J.Inf. Dis. 185: aa 279-287 000-000 (2002); aa 309-317 Elkington, R. etal. J. Virol. 77(9): aa 315-323 5226-5240 (2003). aa 378-389 aa 379-387IE1 Class II aa 91-110 Davignon, J. et al. J. Virol. 70: aa 162-1752162-2169 (1996); aa 96-115 Gautier, N. et al. Eur. J. Immunol. 26(5):1110-7 (1996).

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g. about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

In certain embodiments, the present invention is directed topolynucleotides comprising nucleic acids and fragments thereofcomprising codon-optimized coding regions which encode polypeptides ofHCMV, and in particular, HCMV gB or pp65, and fragments, variants, orderivatives thereof, alone or in combination with additionalcodon-optimized or non-codon-optimized HCMV-derived coding sequences,for example IE1 (SEQ ID NO:19).

“Codon optimization” is defined as modifying a nucleic acid sequence forenhanced expression in the cells of the vertebrate of interest, e.g.human, by replacing at least one, more than one, or a significantnumber, of codons of the native sequence with codons that are morefrequently or most frequently used in the genes of that vertebrate.Various species exhibit particular bias for certain codons of aparticular amino acid.

The present invention relates to polynucleotides comprising nucleic acidfragments of codon-optimized coding regions which encode HCMVpolypeptides, or fragments, variants, or derivatives thereof, with thecodon usage adapted for optimized expression in human cells. Thesepolynucleotides are prepared by incorporating codons preferred for usein human genes into the DNA sequence. Also provided are polynucleotideexpression constructs, vectors, and host cells comprising nucleic acidfragments of codon-optimized coding regions which encode HCMVpolypeptides, and fragments, variants, or derivatives thereof, andvarious methods of using the polynucleotide expression constructs,vectors, host cells to treat or prevent HCMV disease in a human.

Polynucleotides comprising nucleic acid fragments of codon-optimizedcoding regions which encode polypeptides from nonhumancytomegaloviruses, or fragments, variants, or derivatives thereof, maybe optimized for expression in the cells of the vertebrate that can beinfected by the nonhuman cytomegalovirus using the methods describedherein. A partial list of known vertebrate cytomegaloviruses includemurine CMV (MCMV), hamster CMV, guinea pig CMV, rat CMV, rabbit CMV,porcine CMV, bovine CMV, equine CMV, rhesus macaque CMV, African greenmonkey CMV, and chimpanzee CMV, as well as others (Staczek, J., Am. Soc.Microbiol. 545:247-265 (1990)). For example, an MCMV gene would beoptimized for expressing in mouse cells, and an equine CMV gene would beoptimized for expression in horse cells.

Codon Optimization

As used herein the term “codon-optimized coding region” means a nucleicacid coding region that has been adapted for expression in the cells ofa given vertebrate by replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in the genes of that vertebrate.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 2. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 2 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L TCG Ser (S) TAG Ter TGG Trp (W) CCTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro (P)CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R)CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr(T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser(S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACG Thr(T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGTGly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCAAla (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGGGly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available atwww.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). The codon usage table forhuman, calculated from GenBank Release 128.0 [15 Feb. 2002], isreproduced below as Table 3. These tables use mRNA nomenclature, and soinstead of thymine (T) which is found in DNA, the tables use uracil (U)which is found in RNA. The tables have been adapted so that frequenciesare calculated for each amino acid, rather than for all 64 codons. Forcomparison, the codon usage table for human cytomegalovirus isreproduced below as Table 4.

TABLE 3 Codon Usage Table for Human Genes (Homo sapiens) Amino AcidCodon Number Frequency Phe UUU 326146 0.4525 Phe UUC 394680 0.5475 Total720826 Leu UUA 139249 0.0728 Leu UUG 242151 0.1266 Leu CUU 246206 0.1287Leu CUC 374262 0.1956 Leu CUA 133980 0.0700 Leu CUG 777077 0.4062 Total1912925 Ile AUU 303721 0.3554 Ile AUC 414483 0.4850 Ile AUA 1363990.1596 Total 854603 Met AUG 430946 1.0000 Total 430946 Val GUU 2104230.1773 Val GUC 282445 0.2380 Val GUA 134991 0.1137 Val GUG 559044 0.4710Total 1186903 Ser UCU 282407 0.1840 Ser UCC 336349 0.2191 Ser UCA 2259630.1472 Ser UCG 86761 0.0565 Ser AGU 230047 0.1499 Ser AGC 373362 0.2433Total 1534889 Pro CCU 333705 0.2834 Pro CCC 386462 0.3281 Pro CCA 3222200.2736 Pro CCG 135317 0.1149 Total 1177704 Thr ACU 247913 0.2419 Thr ACC371420 0.3624 Thr ACA 285655 0.2787 Thr ACG 120022 0.1171 Total 1025010Ala GCU 360146 0.2637 Ala GCC 551452 0.4037 Ala GCA 308034 0.2255 AlaGCG 146233 0.1071 Total 1365865 Tyr UAU 232240 0.4347 Tyr UAC 3019780.5653 Total 534218 His CAU 201389 0.4113 His CAC 288200 0.5887 Total489589 Gln CAA 227742 0.2541 Gln CAG 668391 0.7459 Total 896133 Asn AAU322271 0.4614 Asn AAC 376210 0.5386 Total 698481 Lys AAA 462660 0.4212Lys AAG 635755 0.5788 Total 1098415 Asp GAU 430744 0.4613 Asp GAC 5029400.5387 Total 933684 Glu GAA 561277 0.4161 Glu GAG 787712 0.5839 Total1348989 Cys UGU 190962 0.4468 Cys UGC 236400 0.5532 Total 427362 Trp UGG248083 1.0000 Total 248083 Arg CGU 90899 0.0830 Arg CGC 210931 0.1927Arg CGA 122555 0.1120 Arg CGG 228970 0.2092 Arg AGA 221221 0.2021 ArgAGG 220119 0.2011 Total 1094695 Gly GGU 209450 0.1632 Gly GGC 4413200.3438 Gly GGA 315726 0.2459 Gly GGG 317263 0.2471 Total 1283759 StopUAA 13963 Stop UAG 10631 Stop UGA 24607

TABLE 4 Codon Usage Table for Human Cytomegalovirus (human herpesvirus5) Amino Acid Codon Number Frequency Phe UUU 5435 0.5456 Phe UUC 45270.4544 Total 9962 Leu UUA 1191 0.0510 Leu UUG 3683 0.1578 Leu CUU 21620.0926 Leu CUC 5473 0.2344 Leu CUA 1771 0.0759 Leu CUG 9066 0.3883 Total23346 Ile AUU 2452 0.2538 Ile AUC 6135 0.6350 Ile AUA 1075 0.1113 Total9662 Met AUG 5051 1.0000 Total 430946 Val GUU 2271 0.1167 Val GUC 50820.2611 Val GUA 2570 0.1320 Val GUG 9541 0.4902 Total 19464 Ser UCU 23500.1234 Ser UCC 3911 0.2054 Ser UCA 1296 0.0681 Ser UCG 4876 0.2561 SerAGU 1927 0.1012 Ser AGC 4677 0.2457 Total 19037 Pro CCU 1817 0.1439 ProCCC 4425 0.3506 Pro CCA 1391 0.1102 Pro CCG 4990 0.3953 Total 12623 ThrACU 2156 0.1368 Thr ACC 5648 0.3584 Thr ACA 1782 0.1131 Thr ACG 61730.3917 Total 15759 Ala GCU 2559 0.1491 Ala GCC 8013 0.4668 Ala GCA 13860.0807 Ala GCG 5209 0.3034 Total 17167 Tyr UAU 2321 0.2629 Tyr UAC 65090.7371 Total 8830 His CAU 1906 0.2753 His CAC 5018 0.7247 Total 6924 GlnCAA 2894 0.3398 Gln CAG 5623 0.6602 Total 8517 Asn AAU 2268 0.2892 AsnAAC 5574 0.7108 Total 7842 Lys AAA 3313 0.4408 Lys AAG 4203 0.5592 Total7516 Asp GAU 3514 0.3023 Asp GAC 8110 0.6977 Total 11624 Glu GAA 43100.3684 Glu GAG 7390 0.6316 Total 11700 Cys UGU 3059 0.4265 Cys UGC 41130.5735 Total 7172 Trp UGG 2797 1.0000 Total 2797 Arg CGU 3747 0.2186 ArgCGC 6349 0.3703 Arg CGA 1826 0.1065 Arg CGG 3285 0.1916 Arg AGA 11850.0691 Arg AGG 752 0.0439 Total 17144 Gly GGU 3521 0.2430 Gly GGC 69520.4797 Gly GGA 1885 0.1301 Gly GGG 2133 0.1472 Total 14491 Stop UAA 310Stop UAG 69 Stop UGA 234

By utilizing these or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons more optimal for a given species.Codon-optimized coding regions can be designed by various differentmethods.

In one method, termed “uniform optimization,” a codon usage table isused to find the single most frequent codon used for any given aminoacid, and that codon is used each time that particular amino acidappears in the polypeptide sequence. For example, referring to Table 3above, for leucine, the most frequent codon is CUG, which is used 41% ofthe time. Thus all the leucine residues in a given amino acid sequencewould be assigned the codon CUG. Human “uniform” codon-optimizednucleotide sequences encoding native pp65 from HCMV strain AD169 (SEQ IDNO:2)) (FIG. 1) and full-length gB from strain AD169 (SEQ ID NO:12)(FIG. 4) are presented herein as SEQ ID NO:7 and SEQ ID NO:15,respectively.

In another method, termed “full-optimization,” the actual frequencies ofthe codons are distributed randomly throughout the coding region. Thus,using this method for optimization, if a hypothetical polypeptidesequence had 100 leucine residues, referring to Table 3 for frequency ofusage in the humans, about 7, or 7% of the leucine codons would be UUA,about 13, or 13% of the leucine codons would be UUG, about 13, or 13% ofthe leucine codons would be CUU, about 20, or 20% of the leucine codonswould be CUC, about 7, or 7% of the leucine codons would be CUA, andabout 41, or 41% of the leucine codons would be CUG. These frequencieswould be distributed randomly throughout the leucine codons in thecoding region encoding the hypothetical polypeptide. As will beunderstood by those of ordinary skill in the art, the distribution ofcodons in the sequence can vary significantly using this method,however, the sequence always encodes the same polypeptide. Threedifferent human codon-optimized nucleotide sequences encoding nativepp65 (SEQ ID NO:2) which have been optimized using this method arepresented herein as SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Threedifferent human codon-optimized sequences encoding native gB (SEQ IDNO:12) which have been fully optimized using this method are presentedherein as SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

In using the “full-optimization” method, an entire polypeptide sequence,or fragment, variant, or derivative thereof is codon-optimized by any ofthe methods described herein. Various desired fragments, variants orderivatives are designed, and each is then codon-optimized individually.Alternatively, a full-length polypeptide sequence is codon-optimized fora given species resulting in a codon-optimized coding region encodingthe entire polypeptide, and then nucleic acid fragments of thecodon-optimized coding region, which encode fragments, variants, andderivatives of the polypeptide are made from the originalcodon-optimized coding region. As would be well understood by those ofordinary skill in the art, if codons have been randomly assigned to thefull-length coding region based on their frequency of use in a givenspecies, nucleic acid fragments encoding fragments, variants, andderivatives would not necessarily be fully codon-optimized for the givenspecies. However, such sequences are still much closer to the codonusage of the desired species than the native codon usage. The advantageof this approach is that synthesizing codon-optimized nucleic acidfragments encoding each fragment, variant, and derivative of a givenpolypeptide, although routine, would be time consuming and would resultin significant expense.

When using the “full-optimization” method, the term “about” is usedprecisely to account for fractional percentages of codon frequencies fora given amino acid. As used herein, “about” is defined as one amino acidmore or one amino acid less than the value given. The whole number valueof amino acids is rounded up if the fractional frequency of usage is0.50 or greater, and is rounded down if the fractional frequency of useis 0.49 or less. Using again the example of the frequency of usage ofleucine in human genes for a hypothetical polypeptide having 62 leucineresidues, the fractional frequency of codon usage would be calculated bymultiplying 62 by the frequencies for the various codons. Thus, 7.28percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUAcodons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e.,7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or“about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percentof 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons,and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e.,24, 25, or 26 CUG codons.

In a third method termed “minimal optimization,” coding regions are onlypartially optimized. For example, the invention includes a nucleic acidfragment of a codon-optimized coding region encoding a polypeptide inwhich at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ofthe codon positions have been codon-optimized for a given species. Thatis, they contain a codon that is preferentially used in the genes of adesired species, e.g., a vertebrate species, e.g., humans, in place of acodon that is normally used in the native nucleic acid sequence. Codonsthat are rarely found in human genes are changed to codons more commonlyutilized in human coding regions. To illustrate this method, acomparative chart showing codon usage per thousand of human and HCMVcoding regions is presented in Table 5. The data is expressed as thenumber of times a given codon is used per 1000 codons. For instance, theasterisked codons in Table 5 for alanine, arginine, proline, serine, andthreonine are frequently used in the genome of HCMV, but less frequentlyused in human genes. Starting with the native coding region of the HCMVgene of interest, one or more codons which are infrequently-used may bechanged to more commonly-used human codons either by substituting one ofthe codons more frequently used in human genes. According to thismethod, these HCMV codons which are used at the same or higher frequencyin human genes as compared to HCMV genes are left unchanged.

TABLE 5 Codon Usage Table for Human Genes and HCMV Amino Acid CodonHuman hCMV Ala A GCA 16 6 * GCG 8 22 GCC 19 34 GCT 19 11 Arg R AGA 12 5AGG 11 3 CGA 6 8 CGG 12 14 CGC 11 27 * CGT 5 16 Asn N AAC 20 24 AAT 1710 Asp D GAC 26 34 GAT 22 15 Cys C TGC 12 17 TGT 10 13 Gln Q CAA 12 12CAG 35 24 Glu E GAA 30 18 GAG 40 31 Gly G GGA 16 8 GGG 16 9 GGC 23 29GGT 11 15 His H CAC 15 21 CAT 11 8 Ile I ATA 7 5 ATC 22 26 ATT 16 10 LeuL CTA 7 8 CTG 40 38 CTC 20 23 CTT 13 9 TTA 7 5 TTG 13 16 Lys K AAA 24 14AAG 33 18 Met M ATG 22 21 Phe F TTC 21 19 TTT 17 23 Pro P CCA 17 6 * CCG7 21 CCC 20 19 CCT 17 8 Ser S AGC 19 20 AGT 12 8 TCA 12 6 * TCG 5 21 TCC18 17 TCT 15 10 Thr T ACA 15 8 * ACG 6 26 ACC 19 24 ACT 13 9 Trp W TGG13 12 Tyr Y TAC 16 27 TAT 12 10 Val V GTA 7 11 GTG 29 40 GTC 15 21 GTT11 10 Term TAA 1 1 TAG 0.5 0 TGA 1 1

Thus, those codons which are used more frequently in the HCMV genomethan in human genes are substituted with the most frequently-used humancodon. The difference in frequency at which the HCMV codons aresubstituted may vary based on a number factors as discussed below. Forexample, codons used at least twice more per thousand in HCMV genes ascompared to human genes are substituted with the most frequently usedhuman codon for that amino acid. This ratio may be adjusted higher orlower depending on various factors such as those discussed below.Accordingly, a codon in an HCMV native coding region would besubstituted with the codon used most frequently for that amino acid inhuman coding regions if the codon is used 1.1 times, 1.2 times, 1.3times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times,2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times,3.3, times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times,4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.5 times, 6.0times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times,9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times,12.5 times, 13.0 times, 13.5 times, 14.0 times, 14.5 times, 15.0 times,15.5 times, 16.0 times, 16.5 times, 17.0 times, 17.5 times, 18.0 times,18.5 times, 19.0 times, 19.5 times, 20 times, 21 times, 22 times, 23times, 24 times, 25 times, or greater more frequently in HCMV codingregions than in human coding regions.

This minimal human codon optimization for highly variant codons hasseveral advantages, which include but are not limited to the followingexamples. Since fewer changes are made to the nucleotide sequence of thegene of interest, fewer manipulations are required, which leads toreduced risk of introducing unwanted mutations and lower cost, as wellas allowing the use of commercially available site-directed mutagenesiskits, reducing the need for expensive oligonucleotide synthesis.Further, decreasing the number of changes in the nucleotide sequencedecreases the potential of altering the secondary structure of thesequence, which can have a significant impact on gene expression incertain host cells. The introduction of undesirable restriction sites isalso reduced, facilitating the subcloning of the genes of interest intothe plasmid expression vector.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTI Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences. For example, the “backtranslation” function isproved on the world wide web by Entelechon GMBH atwww.entelechon.com/eng/backtranslation.html (visited Jul. 9, 2002),“backtranseq” function available atbioinfo.pbi.nrc.ca:-8090/EMBOSS/index.html (visited Oct. 15, 2002).Constructing a rudimentary algorithm to assign codons based on a givenfrequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

A number of options are available for synthesizing codon-optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In one approach, a series ofcomplementary oligonucleotide pairs of 80-90 nucleotides each in lengthand spanning the length of the desired sequence are synthesized bystandard methods. These oligonucleotide pairs are synthesized such thatupon annealing, they form double stranded fragments of 80-90 base pairs,containing cohesive ends, e.g., each oligonucleotide in the pair issynthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond theregion that is complementary to the other oligonucleotide in the pair.The single-stranded ends of each pair of oligonucleotides is designed toanneal with the single-stranded end of another pair of oligonucleotides.The oligonucleotide pairs are allowed to anneal, and approximately fiveto six of these double-stranded fragments are then allowed to annealtogether via the cohesive single stranded ends, and then they ligatedtogether and cloned into a standard bacterial cloning vector, forexample, a TOPO® vector available from Invitrogen Corporation, Carlsbad,Calif. The construct is then sequenced by standard methods. Several ofthese constructs consisting of 5 to 6 fragments of 80 to 90 base pairfragments ligated together, i.e., fragments of about 500 base pairs, areprepared, such that the entire desired sequence is represented in aseries of plasmid constructs. The inserts of these plasmids are then cutwith appropriate restriction enzymes and ligated together to form thefinal construct. The final construct is then cloned into a standardbacterial cloning vector, and sequenced. Additional methods would beimmediately apparent to the skilled artisan. In addition, gene synthesisis readily available commercially.

The codon-optimized coding regions can be versions encoding any geneproducts from any strain of HCMV, or fragments, variants, or derivativesof such gene products. Described herein are nucleic acid fragments ofcodon-optimized coding regions encoding the HCMV pp65 polypeptide andthe HCMV glycoprotein B (gB) polypeptide, the nucleic acid fragmentsencoding the complete polypeptide, as well as various fragments,variants, and derivatives thereof, although other pp65 or gB-encodingnucleic acid sources are not excluded. Codon-optimized coding regionsencoding other HCMV polypeptides (e.g. IE1), or fragments, variants andderivatives thereof, are included within the present invention.Additional, non-codon-optimized polynucleotides encoding HCMVpolypeptides may be included as well.

The present invention is directed to compositions and methods ofenhancing the immune response of a human in need of protection againstHCMV infection by administering in vivo, into a tissue of a human, apolynucleotide comprising a codon-optimized coding region encoding apolypeptide of HCMV, or a nucleic acid fragment of such a coding regionencoding a fragment, variant or derivative thereof. Human-codonoptimization is carried out by the methods described herein, forexample, in certain embodiments codon-optimized coding regions encodingpolypeptides of HCMV, or nucleic acid fragments of such coding regionsencoding fragments, variants, or derivatives thereof are optimizedaccording to human codon usage. The polynucleotides of the invention areincorporated into the cells of the human in vivo, and an immunologicallyeffective amount of an HCMV polypeptide is produced in vivo.

In particular, the present invention relates to codon-optimized codingregions encoding polypeptides of HCMV, or nucleic acid fragments of suchcoding regions fragments, variants, or derivatives thereof which havebeen optimized according to human codon usage. For example, humancodon-optimized coding regions encoding polypeptides of HCMV, or nucleicacid fragments of such coding regions encoding fragments, variants, orderivatives thereof are prepared by substituting one or more codonspreferred for use in human genes for the codons naturally used in theDNA sequence encoding the HCMV polypeptide. Also provided arepolynucleotides, vectors, and other expression constructs comprisingcodon-optimized coding regions encoding polypeptides of HCMV, or nucleicacid fragments of such coding regions encoding fragments, variants, orderivatives thereof, and various methods of using such polynucleotides,vectors and other expression constructs. Coding regions encoding HCMVpolypeptides may be uniformly optimized, fully optimized, or minimallyoptimized, as described herein.

The present invention is further directed towards polynucleotidescomprising codon-optimized coding regions encoding polypeptides of HCMVantigens, for example, HCMV pp65, gB, and optionally in conjunction withother HCMV antigens, e.g. IE1. The invention is also directed topolynucleotides comprising codon-optimized nucleic acid fragmentsencoding fragments, variants and derivatives of these polypeptides.

The present invention provides isolated polynucleotides comprisingcodon-optimized coding regions of HCMV pp65, or fragments, variants, orderivatives thereof. In certain embodiments described herein, acodon-optimized coding region encoding SEQ ID NO:2 is optimizedaccording to codon usage in humans (Homo sapiens).

Codon-optimized coding regions encoding SEQ ID NO:2, fully optimizedaccording to codon usage in humans are designed as follows. The aminoacid composition of SEQ ID NO:2 is shown in Table 6.

TABLE 6 Amino Acid Composition of Wild-Type HCMV pp65 from strain AD169(SEQ ID NO: 2). Number in Amino Acid SEQ ID NO: 2 A Ala 38 R Arg 36 CCys 10 G Gly 36 H His 24 I Ile 25 L Leu 41 K Lys 22 M Met 16 F Phe 19 PPro 38 S Ser 41 T Thr 37 W Trp 9 Y Tyr 15 V Val 44 N Asn 18 D Asp 28 QGln 31 E Glu 33

Using the amino acid composition shown in Table 6, and the human codonusage table shown in Table 3, a human codon-optimized coding regionwhich encodes SEQ ID NO:2 can be designed by any of the methodsdiscussed herein.

In the “uniform optimization” approach, each amino acid is assigned themost frequent codon used in the human genome for that amino acid asindicated on Table 3. According to this method, codons are assigned tothe coding region encoding SEQ ID NO:2 as follows: the 19 phenylalaninecodons are TTC, the 41 leucine codons are CTG, the 25 isoleucine codonsare ATC, the 16 methionine codons are ATG, the 44 valine codons are GTG,the 41 serine codons are AGC, the 38 proline codons are CCC, the 37threonine codons are ACC, the 38 alanine codons are GCC, the 15 tyrosinecodons are TAC, the 24 histidine codons are CAC, the 31 glutamine codonsare CAG, the 18 asparagine codons are AAC, the 22 lysine codons are AAG,the 28 aspartic acid codons are GAC, the 33 glutamic acid codons areGAG, the 10 cysteine codons are TGC, the 9 tryptophan codons are TGG,the 36 arginine codons are CGG, AGA, or AGG (the frequencies of usage ofthese three codons in the human genome are not significantly different),and the 36 glycine codons are GGC. The codon-optimized pp65 codingregion designed by this method is presented herein as SEQ ID NO:7.

Alternatively, a “fully codon-optimized” coding region which encodes SEQID NO:2 can be designed by randomly assigning each of any given aminoacid a codon based on the frequency that codon is used in the humangenome. These frequencies are shown in Table 3 above. Using this lattermethod, codons are assigned to the coding region encoding SEQ ID NO:2 asfollows: about 9 of the 19 phenylalanine codons are TTT, and about 10 ofthe phenylalanine codons are TTC; about 3 of the 41 leucine codons areTTA, about 5 of the leucine codons are TTG, about 5 of the leucinecodons are CTT, about 8 of the leucine codons are CTC, about 3 of theleucine codons are CTA, and about 17 of the leucine codons are CTG;about 9 of the 25 isoleucine codons are ATT, about 12 of the isoleucinecodons are ATC, and about 4 of the isoleucine codons are ATA; the 16methionine codons are ATG; about 8 of the 44 valine codons are GTT,about 10 of the valine codons are GTC, about 5 of the valine codons areGTA, and about 21 of the valine codons are GTG; about 8 of the 41 serinecodons are TCT, about 9 of the serine codons are TCC, about 6 of theserine codons are TCA, about 2 of the serine codons are TCG, about 6 ofthe serine codons are AGT, and about 10 of the serine codons are AGC;about 11 of the 38 proline codons are CCT, about 12 of the prolinecodons are CCC, about 10 of the proline codons are CCA, and about 4 ofthe proline codons are CCG; about 9 of the 37 threonine codons are ACT,about 13 of the threonine codons are ACC, about 11 of the threoninecodons are ACA, and about 4 of the threonine codons are ACG; about 10 ofthe 38 alanine codons are GCT, about 15 of the alanine codons are GCC,about 9 of the alanine codons are GCA, and about 4 of the alanine codonsare GCG; about 7 of the 15 tyrosine codons are TAT and about 8 of thetyrosine codons are TAC; about 10 of the 24 histidine codons are CAT andabout 14 of the histidine codons are CAC; about 8 of the 31 glutaminecodons are CAA and about 23 of the glutamine codons are CAG; about 8 ofthe 18 asparagine codons are AAT and about 10 of the asparagine codonsare AAC; about 9 of the 22 lysine codons are AAA and about 13 of thelysine codons are AAG; about 13 of the 28 aspartic acid codons are GATand about 15 of the aspartic acid codons are GAC; about 14 of the 33glutamic acid codons are GAA and about 19 of the glutamic acid codonsare GAG; about 4 of the 10 cysteine codons are TGU and about 6 of thecysteine codons are TGC; the 9 tryptophan codons are TGG; about 3 of the36 arginine codons are CGT, about 7 of the arginine codons are CGC,about 4 of the arginine codons are CGA, about 8 of the arginine codonsare CGG, about 7 of the arginine codons are AGA, and about 7 of thearginine codons are AGG; and about 6 of the 36 glycine codons are GGT,about 12 of the glycine codons are GGC, about 9 of the glycine codonsare GGA, and about 9 of the glycine codons are GGG.

As described above, the term “about” means that the number of aminoacids encoded by a certain codon may be one more or one less than thenumber given. It would be understood by those of ordinary skill in theart that the total number of any amino acid in the polypeptide sequencemust remain constant, therefore, if there is one “more” of one codonencoding a give amino acid, there would have to be one “less” of anothercodon encoding that same amino acid.

Representative fully-codon-optimized pp65 coding regions designed bythis method are presented herein as SEQ ID NOs:8-10.

Additionally, a minimally codon-optimized nucleotide sequence encodingSEQ ID NO:2 can be designed by changing only certain codons found morefrequently in HCMV genes than in human genes, as shown in Table 5. Forexample, if it is desired to substitute more frequently used codons inhumans for those codons that occur at least 2.7 times more frequently inHCMV genes, Ala CGC, which occurs 2.75 times more frequently in HCMVgenes than in human genes, is changed to, e.g., GCC; Pro CCG, whichoccurs 3.0 times more frequently in HCMV genes than is human, is changedto, e.g., CCC; Arg CGT, which occurs 3.2 times more frequently in HCMVgenes than is human, is changed to, e.g., CGC; Ser TCG, which occurs 4.2times more frequently in HCMV genes than in human, is changed to, e.g.,TCC; and Thr ACG, which occurs 4.3 times more frequently in HCMV genesthan is human, is changed to, e.g., ACC. The minimally codon-optimizedpp65 coding region designed by this method encoding native HCMV pp65 ispresented herein as SEQ ID NO:3. Other methods of “minimal” optimizationcan be carried out by methods well known to those of ordinary skill inthe art.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment which encodes at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 95, or at least 100 or morecontiguous amino acids of SEQ ID NO:2, where the nucleic acid fragmentis a fragment of a codon-optimized coding region encoding SEQ ID NO:2.The human codon-optimized coding region can be optimized by any of themethods described herein.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment which encodes apolypeptide at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO:2, and where the nucleic acid fragment is a variant of a humancodon-optimized coding region encoding SEQ ID NO:2. The humancodon-optimized coding region can be optimized by any of the methodsdescribed herein.

Further provided is an isolated polynucleotide comprising a minimallycodon-optimized nucleic acid (SEQ ID NO:5) which encodes a polypeptidevariant of pp65, i.e., SEQ ID NO:6, in which amino acids 435-438 of SEQID NO:2 have been deleted. This deletion in the amino acid sequence ofpp65 removes putative adventitious substrates for kinase activitypresent in the amino acid sequence. A human codon-optimized codingregion encoding this variant can be optimized by any of the methodsdescribed herein. Alternatively amino acids 435-438 could be substitutedwith different amino acids, or an insertion could be made to remove themotif. Additional fragments, variants, or derivatives of SEQ ID NO:2 maybe utilized as well.

The present invention further provides isolated polynucleotidescomprising human codon-optimized coding regions of HCMV gB, orfragments, variants, or derivatives thereof. In certain embodimentsdescribed herein, a human codon-optimized coding region encoding SEQ IDNO:12 is optimized according to codon usage in humans (Homo sapiens).The human codon-optimized coding region can be optimized by any of themethods described herein.

Codon-optimized coding regions encoding SEQ ID NO:12, optimizedaccording to codon usage in humans are designed as follows. The aminoacid composition of SEQ ID NO:12 is shown in Table 7, and the amino acidcomposition of truncated, secreted gB (SEQ ID NO:14) is shown in Table8.

TABLE 7 Amino Acid Composition of wild typo HCMV gB (SEQ ID NO: 12)Number in Amino Acid SEQ ID NO: 12 A Ala 62 R Arg 53 C Cys 16 G Gly 46 HHis 20 I Ile 48 L Leu 70 K Lys 39 M Met 17 F Phe 34 P Pro 30 S Ser 87 TThr 71 W Trp 8 Y Tyr 51 V Val 71 N Asn 52 D Asp 45 Q Gln 37 E Glu 49

TABLE 8 Amino Acid Composition of secreted HCMV gB, amino acids 1-713(SEQ ID NO: 14) Number in Amino Acid SEQ ID NO: 14 A Ala 41 R Arg 43 CCys 15 G Gly 27 H His 18 I Ile 41 L Leu 51 K Lys 31 M Met 15 F Phe 30 PPro 19 S Ser 73 T Thr 56 W Trp 8 Y Tyr 43 V Val 57 N Asn 44 D Asp 35 QGln 25 E Glu 41

Using the amino acid composition shown in Table 7 and the human codonusage, table shown in Table 3, a human codon-optimized coding regionwhich encodes SEQ ID NO:12 can be designed by any of the methodsdiscussed herein. In the “uniform optimization” approach, each aminoacid is assigned the most frequent codon used in the human genome forthat amino acid as indicated, e.g., in Table 3. According to thismethod, codons are assigned to the coding region encoding SEQ ID NO:12as follows: the 34 phenylalanine codons are TTC, the 70 leucine codonsare CTG, the 48 isoleucine codons are ATC, the 17 methionine codons areATG, the 71 valine codons are GTG, the 87 serine codons are AGC, the 30proline codons are CCC, the 71 threonine codons are ACC, the 62 alaninecodons are GCC, the 51 tyrosine codons are TAC, the 20 histidine codonsare CAC, the 37 glutamine codons are CAG, the 52 asparagine codons areAAC, the 39 lysine codons are AAG, the 45 aspartic acid codons are GAC,the 49 glutamic acid codons are GAG, the 16 cysteine codons are TGC, the8 tryptophan codons are TGG, the 53 arginine codons are CGG, AGA, or AGG(the frequencies of usage of these three codons in the human genome arenot significantly different), and the 46 glycine codons are GGC. Thecodon-optimized full-length gB coding region designed by this method ispresented herein as SEQ ID NO:15.

Alternatively, a “fully codon-optimized” coding region which encodes SEQID NO:12 can be designed by randomly assigning each of any given aminoacid a codon based on the frequency that codon is used in the humangenome. These frequencies are shown in Table 3 above. Using this lattermethod, codons are assigned to the coding region encoding SEQ ID NO:12as follows: about 15 of the 34 phenylalanine codons are TTT and about 19of the phenylalanine codons are TTC; about 5 of the 70 leucine codonsare TTA, about 9 of the leucine codons are TTG, about 9 of the leucinecodons are CTT, about 10 of the leucine codons are CTC, about 5 of theleucine codons are CTA, and about 28 of the leucine codons are CTG;about 17 of the 48 soleucine codons are ATT, about 23 of the isoleucinecodons are ATC, and about 8 of the isoleucine codons are ATA; the 17methionine codons are ATG; about 13 of the 71 valine codons are GTT,about 17 of the valine codons are GTC, about 8 of the valine codons areGTA, and about 33 of the valine codons are GTG; about 16 of the 87serine codons are TCT, about 19 of the serine codons are TCC, about 13of the serine codons are TCA, about 5 of the serine codons are TCG,about 13 of the serine codons are AGT, and about 21 of the serine codonsare AGC; about 9 of the 30 proline codons are CCT, about 10 of theproline codons are CCC, about 8 of the proline codons are CCA, and about3 of the proline codons are CCG; about 17 of the 71 threonine codons areACT, about 26 of the threonine codons are ACC, about 20 of the threoninecodons are ACA, and about 8 of the threonine codons are ACG; about 16 ofthe 62 alanine codons are GCT, about 25 of the alanine codons are GCC,about 14 of the alanine codons are GCA, and about 7 of the alaninecodons are GCG; about 22 of the 51 tyrosine codons are TAT and about 29of the tyrosine codons are TAC; about 8 of the 20 histidine codons areCAT and about 12 of the histidine codons are CAC; about 9 of the 37glutamine codons are CAA and about 28 of the glutamine codons are CAG;about 24 of the 52 asparagine codons are AAT and about 28 of theasparagine codons are AAC; about 16 of the 39 lysine codons are AAA andabout 23 of the lysine codons are AAG; about 21 of the 45 aspartic acidcodons are GAT and about 24 of the aspartic acid codons are GAC; about20 of the 49 glutamic acid codons are GAA and about 29 of the glutamicacid codons are GAG; about 7 of the 16 cysteine codons are TGT and about9 of the cysteine codons are TGC; the 8 tryptophan codons are TOG; about4 of the 53 arginine codons are CGT, about 10 of the arginine codons areCGC, about 6 of the arginine codons are CGA, about 11 of the argininecodons are CGG, about 11 of the arginine codons are AGA, and about 11 ofthe arginine codons are AGG; and about 7 of the 46 glycine codons areGGT, about 16 of the glycine codons are GGC, about 12 of the glycinecodons are GGA, and about 11 of the glycine codons are GGG.

As described above, the term “about” means that the number of aminoacids encoded by a certain codon may be one more or one less than thenumber given. It would be understood by those of ordinary skill in theart that the total number of any amino acid in the polypeptide sequencemust remain constant, therefore, if there is one “more” of one codonencoding a give amino acid, there would have to be one “less” of anothercodon encoding that same amino acid. Representative fullycodon-optimized gB coding regions designed by this method encodingfull-length HCMV gB are presented herein as SEQ ID NOs:16-18.

Additionally, a minimally codon-optimized nucleotide sequence encodingSEQ ID NO:14 can be designed by referring to the amino acid compositionof Table 8 and changing only certain codons found more frequently inhighly expressing human genes, as shown in Table 5. For example, if itis desired to substitute more frequently used codons in humans for thosecodons that occur at least 2.7 times more frequently in HCMV genes, AlaCGC, which occurs 2.75 times more frequently in HCMV genes than in humangenes, is changed to, e.g., GCC; Pro CCG, which occurs 3.0 times morefrequently in HCMV genes than is human, is changed to, e.g., CCC; ArgCGT, which occurs 3.2 times more frequently in HCMV genes than is human,is changed to, e.g., CGC; Ser TCG, which occurs 4.2 times morefrequently in HCMV genes than in human, is changed to, e.g., TCC; andThr ACG, which occurs 4.3 times more frequently in HCMV genes than ishuman, is changed to, e.g., ACC. The minimally codon-optimized secretedgB coding region encoding SEQ ID NO:14 designed by this method ispresented herein as SEQ ID NO:13.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment which encodes at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 95, or at least 100 or morecontiguous amino acids of SEQ ID NO:12 or SEQ ID NO:14, where thenucleic acid fragment is a fragment of a human codon-optimized codingregion encoding SEQ ID NO:12 or SEQ ID NO:14. The human codon-optimizedcoding region can be optimized by any of the methods described herein.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid which encodes a polypeptide atleast 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to gB, i.e., SEQ ID NO:12or SEQ ID NO:14, and where the nucleic acid is a variant of acodon-optimized coding region encoding SEQ ID NO:12 or SEQ ID NO:14. Thehuman codon-optimized coding region can be optimized by any of themethods described herein.

In this manner, the present invention provides a method of enhancing thelevel of polypeptide expression from delivered polynucleotides in vivoand/or facilitating uptake of the polynucleotides by the cells of adesired species, for example a vertebrate species, for example amammalian species, for example humans. Accordingly, the presentinvention provides a method of treatment and prevention against HCMVinfection.

Methods and Administration

The present invention further provides methods for delivering an HCMVpolypeptide to a human, which comprise administering to a human one ormore of the compositions described herein; such that upon administrationof compositions such as those described herein, an HCMV polypeptide isexpressed in human cells, in an amount sufficient generate an immuneresponse to HCMV.

The term “vertebrate” is intended to encompass a singular “vertebrate”as well as plural “vertebrates” and comprises mammals and birds, as wellas fish, reptiles, and amphibians.

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys, orangutans, and chimpanzees; canids such as dogsand wolves; felids such as cats, lions, and tigers; equines such ashorses, donkeys, and zebras, food animals such as cows, pigs, and sheep;ungulates such as deer and giraffes; and ursids such as bears. Inparticular, the mammal can be a human subject, a food animal or acompanion animal.

The present invention further provides a method for generating,enhancing or modulating an immune response to HCMV comprisingadministering to a vertebrate one or more of the compositions describedherein. In this method, the composition includes an isolatedpolynucleotide comprising a human codon-optimized coding region encodinga polypeptide of HCMV, or a nucleic acid fragment of such a codingregion encoding a fragment, variant, or derivative thereof. Thepolynucleotides are incorporated into the cells of the vertebrate invivo, and an antigenic amount of the HCMVs polypeptide, or fragment,variant, or derivative thereof, is produced in vivo. Upon administrationof the composition according to this method, the HCMV polypeptide isexpressed in the vertebrate in an amount sufficient to elicit an immuneresponse. Such an immune response might be used, for example, togenerate antibodies to HCMV for use in diagnostic assays or aslaboratory reagents.

The present invention further provides a method for generating,enhancing, or modulating a protective and/or therapeutic immune responseto HCMV in a human, comprising administering to a human in need oftherapeutic and/or preventative immunity one or more of the compositionsdescribed herein. In this method, the composition includes an isolatedpolynucleotide comprising a human codon-optimized coding region encodinga polypeptide of HCMV, or a nucleic acid fragment of such a codingregion encoding a fragment, variant, or derivative thereof. Thepolynucleotides are incorporated into the cells of the human in vivo,and an immunologically effective amount of the HCMV polypeptide, orfragment or variant is produced in vivo. Upon administration of thecomposition according to this method, the HCMV polypeptide is expressedin the human in a therapeutically or prophylactically effective amount.

As used herein, an “immune response” refers to the ability of avertebrate to elicit an immune reaction to a composition delivered tothat vertebrate. Examples of immune responses include an antibodyresponse or a cellular, e.g., cytotoxic T-cell, response. One or morecompositions of the present invention may be used to prevent HCMVinfection in humans, e.g., as a prophylactic vaccine, to establish orenhance immunity to HCMV in a healthy individual prior to exposure toHCMV or contraction of HCMV disease, thus preventing the disease orreducing the severity of disease symptoms.

One or more compositions of the present invention may also be used totreat individuals already exposed to HCMV, or already suffering fromHCMV disease to further stimulate the immune system of the human, thusreducing or eliminating the symptoms associated with that disease ordisorder. As defined herein, “treatment” refers to the use of one ormore compositions of the present invention to prevent, cure, retard, orreduce the severity of HCMV disease symptoms in a human, and/or resultin no worsening of HCMV disease over a specified period of time. It isnot required that any composition of the present invention provide totalimmunity to HCMV or totally cure or eliminate all HCMV disease symptoms.As used herein, a “human in need of therapeutic and/or preventativeimmunity” refers to an individual for whom it is desirable to treat,i.e., to prevent, cure, retard, or reduce the severity of HCMV diseasesymptoms, and/or result in no worsening of HCMV disease over a specifiedperiod of time.

In other embodiments, one or more compositions of the present inventionare utilized in a “prime boost” regimen. An example of a “prime boost”regimen may be found in Yang; Z. et al. J. Virol. 77:799-803 (2002). Inthese embodiments, one or more polynucleotide vaccine compositions ofthe present invention are delivered to a human, thereby priming theimmune response of the human to HCMV, and then a second immunogeniccomposition is utilized as a boost vaccination. One or morepolynucleotide vaccine compositions of the present invention are used toprime immunity, and then a second immunogenic composition, e.g., arecombinant viral vaccine or vaccines, a different polynucleotidevaccine, one or more purified subunit HCMV proteins, e.g., gB or pp65,with or without additional HCMV antigens, e.g. IE1, or a variant,fragment, or derivative thereof, is used to boost the anti-HCMV immuneresponse. The polynucleotide vaccine compositions may comprise one ormore vectors for expression of one or more HCMV genes as describedherein. In addition, a polynucleotide prime vaccine and the later boostvaccine may elicit an immune response to the same or similar antigens,or may elicit responses to different antigens.

In another embodiment, vectors are prepared for expression in therecombinant virus vaccine and in transfected mammalian cells as part ofa polynucleotide vaccine.

The terms “priming” or “primary” and “boost” or “boosting” are usedherein to refer to the initial and subsequent immunizations,respectively, i.e., in accordance with the definitions these termsnormally have in immunology.

The invention further provides methods for enhancing the immune responseof a human patient to HCMV by administering to the tissues of a humanone or more polynucleotides comprising one or more codon-optimizedcoding regions encoding polypeptides of HCMV, and also HCMV polypeptidesor fragments, variants or derivatives thereof; or one or morenon-optimized polynucleotides encoding HCMV polypeptides, fragments,variants or derivatives thereof.

The combination of HCMV polypeptides or polynucleotides encoding HCMVpolypeptides or fragments, variants or derivatives thereof, with thecodon-optimized nucleic acid compositions provides for therapeuticallybeneficial effects at dose sparing concentrations. For example,immunological responses sufficient for a therapeutically beneficialeffect may be attained by using less of a conventional-type vaccine(that is a vaccine comprising immunogenic polypeptides or nucleotidesencoding immunogenic polypeptides, fragments, variants, or derivativesthereof, that are not products of, or have not been codon-optimized asdescribed herein) when supplemented or enhanced with the appropriateamount of a codon-optimized nucleic acid.

Conventional-type vaccines, include vaccine compositions comprisingeither dead, inert or fragments of a virus or bacteria, or bacterial orviral proteins or protein fragments, injected into the patient to elicitaction by the immune system. With regard to the present invention,conventional-type vaccines include compositions comprising immunogenicpolypeptides or nucleotides encoding immunogenic polypeptides,fragments, variants, or derivatives thereof, and vectors comprisingnucleotides encoding immunogenic polypeptides, fragments, variants, orderivatives thereof, that are not products of, or do not containcodon-optimized polynucleotides as described herein. Thus, geneticallyengineered vaccines, are included in conventional-type vaccines, such asgenetically engineered live vaccines, live chimeric vaccines, livereplication-defective vaccines, subunit vaccines, peptide vaccines invarious modifications of monovalent, multivalent, or chimeric subunitvaccines delivered as individual components or incorporated intovirus-like particles for improved immunogenicity, and polynucleotidevaccines. Auxiliary agents, as described herein, are also consideredcomponents of conventional-type vaccines.

Thus, dose sparing is contemplated by administration of thecombinatorial polynucleotide-vaccine compositions of the presentinvention.

In particular, the dose of conventional-type vaccines may be reduced byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered in combinationwith the codon-optimized nucleic acid compositions of the invention.

Similarly, a desirable level of an immunological response afforded by aDNA based pharmaceutical alone may be attained with less DNA byincluding a conventional-type DNA vaccine. Further, using a combinationof a conventional-type vaccine and a codon-optimized DNA-based vaccinemay allow both materials to be used in lesser amounts while stillaffording the desired level of immune response arising fromadministration of either component alone in higher amounts (e.g. one mayuse less of either immunological product when they are used incombination). This reduction in amounts of materials being delivered maybe for each administration, in addition to reducing the number ofadministrations, in a vaccination regimen (e.g. 2 versus 3 or 4injections). Further, the combination may also provide for reducing thekinetics of the immunological response (e.g. desired response levels areattained in 3 weeks instead of 6 after immunization).

In particular, the dose of DNA based pharmaceuticals, may be reduced byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or at least 70% when administered in combinationwith conventional IV vaccines.

Determining the precise amounts of DNA based pharmaceutical and aconventional antigen is based on a number of factors as describedherein, and is readily determined by one of ordinary skill in the art.

In addition to dose sparing, the claimed combinatorial compositionsprovide for a broadening of the immune response and/or enhancedbeneficial immune responses. Such broadened or enhanced immune responsesare achieved by: adding DNA to enhance cellular responses to aconventional-type vaccine; adding a conventional-type vaccine to a DNApharmaceutical to enhanced humoral response; using a combination thatinduces additional epitopes (both humoral and/or cellular) to berecognized and/or more desirably responded to (epitope broadening);employing a DNA-conventional vaccine combination designed for aparticular desired spectrum of immunological responses; obtaining adesirable spectrum by using higher amounts of either component. Thebroadened immune response is measurable by one of ordinary skill in theart by standard immunological assay specific for the desirable responsespectrum.

Both broadening and dose sparing may be obtained simultaneously.

In certain embodiments, one or more compositions of the presentinvention are delivered to a human by methods described herein, therebyachieving an effective therapeutic and/or an effective preventativeimmune response.

More specifically, the compositions of the present invention may beadministered to any tissue of a human, including, but not limited to,muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue,bone marrow tissue, thymus tissue, heart tissue, e.g., myocardium,endocardium, and pericardium, lymph tissue, blood tissue, bone tissue,pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue,intestinal tissue, testicular tissue, ovarian tissue, uterine tissue,vaginal tissue, rectal tissue, nervous system tissue, eye tissue,glandular tissue, tongue tissue, and connective tissue, e.g., cartilage.

Furthermore, the compositions of the present invention may beadministered to any internal cavity of a human, including, but notlimited to, the lungs, the mouth, the nasal cavity, the stomach, theperitoneal cavity, the intestine, any heart chamber, veins, arteries,capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity,the rectal cavity, joint cavities, ventricles in brain, spinal canal inspinal cord, the ocular cavities, the lumen of a duct of a salivarygland or a liver. When the compositions of the present invention isadministered to the lumen of a duct of a salivary gland or liver, thedesired polypeptide is expressed in the salivary gland and the liversuch that the polypeptide is delivered into the blood stream of thehuman from each of the salivary gland or the liver. Certain modes foradministration to secretory organs of a gastrointestinal system usingthe salivary gland, liver and pancreas to release a desired polypeptideinto the bloodstream is disclosed in U.S. Pat. Nos. 5,837,693 and6,004,944, both of which are incorporated herein by reference in theirentireties.

In one embodiment, the compositions are administered to muscle, eitherskeletal muscle or cardiac muscle, or to lung tissue. Specific, butnon-limiting modes for administration to lung tissue are disclosed inWheeler, C. J., et al., Proc. Natl. Acad. Sci. USA 93:11454-11459(1996), which is incorporated herein by reference in its entirety.

According to the disclosed methods, compositions of the presentinvention can be administered by intramuscular (i.m.), subcutaneous(s.c.), or intrapulmonary routes. Other suitable routes ofadministration include, but not limited to intratracheal, transdermal,intraocular, intranasal, inhalation, intracavity, intravenous (i.v.),intraductal (e.g., into the pancreas) and intraparenchymal (i.e., intoany tissue) administration. Transdermal delivery includes, but notlimited to intradermal (e.g., into the dermis or epidermis), transdermal(e.g., percutaneous) and transmucosal administration (i.e., into orthrough skin or mucosal tissue). Intracavity administration includes,but not limited to administration into oral, vaginal, rectal; nasal,peritoneal, or intestinal cavities as well as, intrathecal (i.e., intospinal canal), intraventricular (i.e., into the brain ventricles or theheart ventricles), intraatrial (i.e., into the heart atrium) and subarachnoid (i.e., into the sub arachnoid spaces of the brain)administration.

Any mode of administration can be used so long as the mode results inthe expression of the desired peptide or protein, in the desired tissue,in an amount sufficient to generate an immune response to HCMV and/or togenerate a prophylactically or therapeutically effective immune responseto HCMV in a human in need of such response. Administration means of thepresent invention include needle injection, catheter infusion, biolisticinjectors, particle accelerators (e.g., “gene guns” or pneumatic“needleless” injectors) Med-E-Jet (Vahlsing, H., et al., J. Immunol.Methods 171:11-22 (1994)), Pigjet (Schrijver, R., et al., Vaccine 15:1908-1916 (1997)), Biojector (Davis, H., et al., Vaccine 12: 1503-1509(1994); Gramzinski, R., et al., Mol. Med. 4: 109-118 (1998)), Advantajet(Linmayer, I., et al., Diabetes Care 9:294-297 (1986)), Medi-jector(Martins, J., and Roedl, E. J. Occup. Med. 21:821-824 (1979)), gelfoamsponge depots, other commercially available depot materials (e.g.,hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, topical skin creams,and decanting, use of polynucleotide coated suture (Qin, Y., et al.,Life Sciences 65: 2193-2203 (1999)) or topical applications duringsurgery. Certain modes of administration are intramuscular needle-basedinjection and pulmonary application via catheter infusion.Energy-assisted plasmid delivery (EAPD) methods may also be employed toadminister the compositions of the invention. One such method involvesthe application of brief electrical pulses to injected tissues, aprocedure commonly known as electroporation. See generally Mir, L. M. etal., Proc. Natl. Acad. Sci USA 96:4262-7 (1999); Hartikka, J. et al.,Mol. Ther. 4:407-15 (2001); Mathiesen, I., Gene Ther. 6:508-14 (1999);Rizzuto G. et al., Hum. Gen. Ther. 11:1891-900 (2000). Each of thereferences cited in this paragraph is incorporated herein by referencein its entirety.

Further, antigen constructs alone or in combination may be formulated toenhance the type of immune response (e.g. humoral, cellular, mucosal,etc.) believed to be most beneficial to mount in the host for thatparticular antigen or antigens. Each such formulation may beadministered individually at a separate site in the host, and/orcombined and administered with some or all of the other antigenformulations at one or more sites in the host. Each administration maybe accomplished using the same or different physical means ofadministration. Thus, as a non-limiting example, a gB plasmid could beformulated with cationic lipids and administered as a mist intranasaly,in conjunction with administration of a poloxamer formulation of pp65using a needle free device into skin and muscle of one limb, inconjunction with trans-dermal intramuscular administration using aconventional syringe and needle of an IE1 plasmid in PBS into a secondlimb.

Determining an effective amount of one or more compositions of thepresent invention depends upon a number of factors including, forexample, the antigen being expressed, e.g. gB, pp65 or IE1; orfragments, variants, or derivatives thereof, the age and weight of thesubject, the precise condition requiring treatment and its severity, andthe route of administration. Based on the above factors, determining theprecise amount, number of doses, and timing of doses are within theordinary skill in the art and will be readily determined by theattending physician.

Compositions of the present invention may include various salts,excipients, delivery vehicles and/or auxiliary agents as are disclosed,e.g., in U.S. Patent Application Publication 2002/0019358, publishedFeb. 14, 2002, which is incorporated herein by reference in itsentirety.

Furthermore, compositions of the present invention may include one ormore transfection facilitating compounds that facilitate delivery ofpolynucleotides to the interior of a cell, and/or to a desired locationwithin a cell. As used herein, the terms “transfection facilitatingcompound,” “transfection facilitating agent,” and “transfectionfacilitating material” are synonymous, and may be used interchangeably.It should be noted that certain transfection facilitating compounds mayalso be “adjuvants” as described infra, e.g., in addition tofacilitating delivery of polynucleotides to the interior of a cell, thecompound acts to alter or increase the immune response to the antigenencoded by that polynucleotide. Examples of the transfectionfacilitating compounds include, but are not limited to inorganicmaterials such as calcium phosphate, alum (aluminum sulfate), and goldparticles (e.g., “powder” type delivery vehicles); peptides that are,for example, cationic, intercell targeting (for selective delivery tocertain cell types), intracell targeting (for nuclear localization orendosomal escape), and ampipathic (helix forming or pore forming);proteins that are, for example, basic (e.g., positively charged) such ashistones, targeting (e.g., asialoprotein), viral (e.g., Sendai viruscoat protein), and pore-forming; lipids that are, for example, cationic(e.g., DMRIE, DOSPA, DC-Chol), basic (e.g., steryl amine), neutral(e.g., cholesterol), anionic (e.g., phosphatidyl serine), andzwitterionic (e.g., DOPE, DOPC); and polymers such as dendrimers,star-polymers, “homogenous” poly-amino acids (e.g., poly-lysine,poly-arginine), “heterogenous” poly amino acids (e.g., mixtures oflysine & glycine), co-polymers, polyvinylpyrrolidinone (PVP), poloxamers(e.g. CRL 1005) and polyethylene glycol (PEG). A transfectionfacilitating material can be used alone or in combination with one ormore other transfection facilitating materials. Two or more transfectionfacilitating materials can be combined by chemical bonding (e.g.,covalent and ionic such as in lipidated polylysine, PEGylatedpolylysine) (Toncheva, et al., Biochim. Biophys. Acta 1380(3):354-368(1988)), mechanical mixing (e.g., free moving materials in liquid orsolid phase such as “polylysine+cationic lipids”) (Gao and Huang,Biochemistry 35:1027-1036 (1996); Trubetskoy, et al., Biochem. Biophys.Acta 1131:311-313 (1992)), and aggregation (e.g., co-precipitation, gelforming such as in cationic lipids+poly-lactide, andpolylysine+gelatin).

One category of transfection facilitating materials is cationic lipids.Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide(DOGS) and dipalmitoyl-phophatidylethanolamine-5-carboxyspermylamide(DPPES). Cationic cholesterol derivatives are also useful, including{3β-[N—N′,N′-dimethylamino)ethane]-carbomoyl}-cholesterol(DC-Chol).Dimethyldioctdecyl-ammonium bromide (DDAB),N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammoniumbromide (PA-DEMO),N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammoniumbromide (PA-DELO),N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide(PA-TELO), andN1-(3-aminopropyl)((2-dodecyloxy)ethyl)-N2-(2-dodecyloxy)ethyl-1-piperazinaminiumbromide (GA-LOE-BP) can also be employed in the present invention.

Non-diether cationic lipids, such asDL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORIdiester),1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORIester/ether), and their salts promote in vivo gene delivery. In someembodiments, cationic lipids comprise groups attached via a heteroatomattached to the quaternary ammonium moiety in the head group. A glycylspacer can connect the linker to the hydroxyl group.

Specific, but non-limiting cationic lipids for use in certainembodiments of the present invention include DMRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide), GAP-DMORIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide), and GAP-DLRIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanaminiumbromide).

Other specific but non-limiting cationic surfactants for use in certainembodiments of the present invention include Bn-DHRIE, DhxRIE,DhxRIE-OAc, DhxRIE-OBz and Pr-DOctRIE-OAc. These lipids are disclosed incopending U.S. Patent Application Ser. No. 60/435,303. In another aspectof the present invention, the cationic surfactant is Pr-DOctRIE-OAc.

Other cationic lipids include(±)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)-1-propaniminiumpentahydrochloride (DOSPA),(±)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminiumbromide(β-aminoethyl-DMRIE or RAE-DMRIE) (Wheeler, et al., Biochim.Biophys. Acta 1280:1-11 (1996)), and(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminiumbromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA93:11454-11459 (1996)), which have been developed from DMRIE.

Other examples of DMRIE-derived cationic lipids that are useful for thepresent invention are(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminiumbromide (GAP-DDRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanaminiumbromide (GAP-DMRIE),(±)-N—((N″-methyl)-N′-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (GMU-DMRIE),(±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (DLRIE), and(±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)propyl-1-propaniminiumbromide (HP-DORIE).

In the embodiments where the immunogenic composition comprises acationic lipid, the cationic lipid may be mixed with one or moreco-lipids. For purposes of definition, the term Aco-lipid refers to anyhydrophobic material which may be combined with the cationic lipidcomponent and includes amphipathic lipids, such as phospholipids, andneutral lipids, such as cholesterol. Cationic lipids and co-lipids maybe mixed or combined in a number of ways to produce a variety ofnon-covalently bonded macroscopic structures, including, for example,liposomes, multilamellar vesicles, unilamellar vesicles, micelles, andsimple films. One non-limiting class of co-lipids are the zwitterionicphospholipids, which include the phosphatidylethanolamines and thephosphatidylcholines. Examples of phosphatidylethanolamines, includeDOPE, DMPE and DPyPE. In certain embodiments, the co-lipid is DPyPE,which comprises two phytanoyl substituents incorporated into thediacylphosphatidylethanolamine skeleton. In other embodiments, theco-lipid is DOPE, CAS name1,2-diolyeoyl-sn-glycero-3-phosphoethanolamine.

When a composition of the present invention comprises a cationic lipidand co-lipid, the cationic lipid:co-lipid molar ratio may be from about9:1 to about 1:9, from about 4:1 to about 1:4, from about 2:1 to about1:2, or about 1:1.

In order to maximize homogeneity, the plasmid and co-lipid componentsmay be dissolved in a solvent such as chloroform, followed byevaporation of the cationic lipid/co-lipid solution under vacuum todryness as a film on the inner surface of a glass vessel (e.g., aRotovap round-bottomed flask). Upon suspension in an aqueous solvent,the amphipathic lipid component molecules self-assemble into homogenouslipid vesicles. These lipid vesicles may subsequently be processed tohave a selected mean diameter of uniform size prior to complexing with,for example, a codon-optimized polynucleotide of the present invention,according to methods known to those skilled in the art. For example, thesonication of a lipid solution is described in Felgner et al., Proc.Natl. Acad. Sci. USA 8:, 7413-7417 (1987) and in U.S. Pat. No.5,264,618, the disclosures of which are incorporated herein byreference.

In those embodiments where the composition includes a cationic lipid,polynucleotides of the present invention are complexed with lipids bymixing, for example, a plasmid in aqueous solution and a solution ofcationic lipid:co-lipid as prepared herein are mixed. The concentrationof each of the constituent solutions can be adjusted prior to mixingsuch that the desired final plasmid/cationic lipid:co-lipid ratio andthe desired plasmid final concentration will be obtained upon mixing thetwo solutions. The cationic lipid:co-lipid mixtures are suitablyprepared by hydrating a thin film of the mixed lipid materials in anappropriate volume of aqueous solvent by vortex mixing at ambienttemperatures for about 1 minute. The thin films are prepared by admixingchloroform solutions of the individual components to afford a desiredmolar solute ratio followed by aliquoting the desired volume of thesolutions into a suitable container. The solvent is removed byevaporation, first with a stream of dry, inert gas (e.g. argon) followedby high vacuum treatment.

Other hydrophobic and amphiphilic additives, such as, for example,sterols, fatty acids, gangliosides, glycolipids, lipopeptides,liposaccharides, neobees, niosomes, prostaglandins and sphingolipids,may also be included in compositions of the present invention. In suchcompositions, these additives may be included in an amount between about0.1 mol % and about 99.9 mol % (relative to total lipid), about 1-50 mol%, or about 2-25 mol %.

Additional embodiments of the present invention are drawn tocompositions comprising an auxiliary agent which is administered before,after, or concurrently with the polynucleotide. As used herein, an“auxiliary agent” is a substance included in a composition for itsability to enhance, relative to a composition which is identical exceptfor the inclusion of the auxiliary agent, the entry of polynucleotidesinto vertebrate cells in vivo, and/or the in vivo expression ofpolypeptides encoded by such polynucleotides. Certain auxiliary agentsmay, in addition to enhancing entry of polynucleotides into cells,enhance an immune response to an immunogen encoded by thepolynucleotide. Auxiliary agents of the present invention includenonionic, anionic, cationic, or zwitterionic surfactants or detergents;chelators, DNAse inhibitors, poloxamers, agents that aggregate orcondense nucleic acids, emulsifying or solubilizing agents, wettingagents, gel-forming agents, and buffers.

Auxiliary agents for use in compositions of the present inventioninclude, but are not limited to non-ionic detergents and surfactantsIGEPAL CA 630® CA 630, NONIDET® NP-40, NONIDET® P40(2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxyl]ethoxyl]ethanol),TWEEN-20™(2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyldodecanoate), TWEEN-80™, Pluronic® F68, Pluronic® F77, Pluronic® P65,Triton X-100™, and Triton X-114™; the anionic detergent sodium dodecylsulfate (SDS); the sugar stachyose; the condensing agent DMSO; and thechelator/DNAse inhibitor EDTA, CRL 1005, and BAK. In certain specificembodiments, the auxiliary agent is DMSO, NONIDET® P40(2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol), Pluronic®F68, Pluronic® F77, Pluronic® P65, Pluronic® L64, and Pluronic® F108.See, e.g., U.S. Patent Application Publication 20020019358, publishedFeb. 14, 2002, which is incorporated herein by reference in itsentirety.

Certain compositions of the present invention may further include one ormore adjuvants before, after, or concurrently with the polynucleotide.The term “adjuvant” refers to any material having the ability to (1)alter or increase the immune response to a particular antigen or (2)increase or aid an effect of a pharmacological agent. It should benoted, with respect to polynucleotide vaccines, that an “adjuvant,” maybe a transfection facilitating material. Similarly, certain“transfection facilitating materials” described supra, may also be an“adjuvant.” An adjuvant may be used with a composition comprising apolynucleotide of the present invention. In a prime-boost regimen, asdescribed herein, an adjuvant may be used with either the primingimmunization, the booster immunization, or both. Suitable adjuvantsinclude, but are not limited to, cytokines and growth factors; bacterialcomponents (e.g., endotoxins, in particular superantigens, exotoxins andcell wall components); aluminum-based salts; calcium-based salts;silica; polynucleotides; toxoids; serum proteins, viruses andvirally-derived materials, poisons, venoms, poloxamers, and cationiclipids.

A great variety of materials have been shown to have adjuvant activitythrough a variety of mechanisms. Any compound which may increase theexpression, antigenicity or immunogenicity of the polypeptide is apotential adjuvant. The present invention provides an assay to screenfor improved immune responses to potential adjuvants. Potentialadjuvants which may be screened for their ability to enhance the immuneresponse according to the present invention include, but are not limitedto: inert carriers, such as alum, bentonite, latex, and acrylicparticles; pluronic block polymers, such as TiterMax™; depot formers,such as Freunds adjuvant, surface active materials, such as saponin,lysolecithin, retinal, Quil A, liposomes, and pluronic polymerformulations; macrophage stimulators, such as bacteriallipopolysaccharide; alternate pathway complement activators; such asinsulin, zymosan, endotoxin, and levamisole; and non-ionic surfactants,such as poloxamers, poly(oxyethylene)-poly(oxypropylene)tri-blockcopolymers. Also included as adjuvants are transfection-facilitatingmaterials, such as those described above.

Poloxamers which may be screened for their ability to enhance the immuneresponse according to the present invention include, but are not limitedto commercially available poloxamers such as Pluronic® L121 (ave. MW:4400; approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 10%),Pluronic® L101 (ave. MW: 3800; approx. MW of hydrophobe, 3000; approx.wt. % of hydrophile, 10%), Pluronic® L81 (ave. MW: 2750; approx. MW ofhydrophobe, 2400; approx. wt. % of hydrophile, 10%), Pluronic® L61 (ave.MW: 2000; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,10%), Pluronic® L31 (ave. MW: 1100; approx. MW of hydrophobe, 900;approx. wt. % of hydrophile, 10%), Pluronic® L122 (ave. MW: 5000;approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 20%),Pluronic® L92 (ave. MW: 3650; approx. MW of hydrophobe, 2700; approx.wt. % of hydrophile; 20%), Pluronic® L72 (ave. MW: 2750; approx. MW ofhydrophobe, 2100; approx. wt. % of hydrophile, 20%), Pluronic® L62 (ave.MW: 2500; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,20%), Pluronic® L42 (ave. MW: 1630; approx. MW of hydrophobe, 1200;approx. wt. % of hydrophile, 20%), Pluronic® L63 (ave. MW: 2650; approx.MW of hydrophobe, 1800; approx. wt. % of hydrophile, 30%), Pluronic® L43(ave. MW: 1850; approx. MW of hydrophobe, 1200; approx. wt. % ofhydrophile, 30%), Pluronic® L64 (ave. MW: 2900; approx. MW ofhydrophobe, 1800; approx. wt. % of hydrophile, 40%), Pluronic® L44 (ave.MW: 2200; approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile,40%), Pluronic® L35 (ave. MW: 1900; approx. MW of hydrophobe, 900;approx. wt. % of hydrophile, 50%), Pluronic® P123 (ave. MW: 5750;approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 30%),Pluronic® P103 (ave. MW: 4950; approx. MW of hydrophobe, 3000; approx.wt. % of hydrophile, 30%), Pluronic® P104 (ave. MW: 5900; approx. MW ofhydrophobe, 3000; approx. wt. % of hydrophile, 40%), Pluronic® P84 (ave.MW: 4200; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile,40%), Pluronic® P105 (ave. MW: 6500; approx. MW of hydrophobe, 3000;approx. wt. % of hydrophile, 50%), Pluronic® P85 (ave. MW: 4600; approx.MW of hydrophobe, 2400; approx. wt. % of hydrophile, 50%), Pluronic® P75(ave. MW: 4150; approx. MW of hydrophobe, 2100; approx. wt. % ofhydrophile, 50%), Pluronic® P65 (ave. MW: 3400; approx. MW ofhydrophobe, 1800; approx. wt. % of hydrophile, 50%), Pluronic® F127(ave. MW: 12600; approx. MW of hydrophobe, 3600; approx. wt. % ofhydrophile, 70%), Pluronic® F98 (ave. MW: 13000; approx. MW ofhydrophobe, 2700; approx. wt. % of hydrophile, 80%), Pluronic® F87 (ave.MW: 7700; approx. MW of hydrophobe, 0.2400; approx. wt. % of hydrophile,70%), Pluronic® F77 (ave. MW: 6600; approx. MW of hydrophobe, 2100;approx. wt. % of hydrophile, 70%), Pluronic® F108 (ave. MW: 14600;approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 80%),Pluronic® F98 (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx.wt. % of hydrophile, 80%), Pluronic® F88 (ave. MW: 11400; approx. MW ofhydrophobe, 2400; approx. wt. % of hydrophile, 80%), Pluronic® F68 (ave.MW: 8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,80%), Pluronic® F38 (ave. MW: 4700; approx. MW of hydrophobe, 900;approx. wt. % of hydrophile, 80%).

Reverse poloxamers of the present invention include, but are not limitedto Pluronic® R 31R1 (ave. MW: 3250; approx. MW of hydrophobe, 3100;approx. wt. % of hydrophile, 10%), Pluronic® R 25R1 (ave. MW: 2700;approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 10%),Pluronic® R 17R1 (ave. MW: 1900; approx. MW of hydrophobe, 1700; approx.wt. % of hydrophile, 10%), Pluronic® R 31R2 (ave. MW: 3300; approx. MWof hydrophobe, 3100; approx. wt: % of hydrophile, 20%), Pluronic® R 25R2(ave. MW: 3100; approx. MW of hydrophobe, 2500; approx. wt. % ofhydrophile, 20%), Pluronic® R 17R2 (ave. MW: 2150; approx. MW ofhydrophobe, 1.700; approx. wt. % of hydrophile, 20%), Pluronic® R 12R3(ave. MW: 1800; approx. MW of hydrophobe, 1200; approx. wt. % ofhydrophile, 30%), Pluronic® R 31R4 (ave. MW: 4150; approx. MW ofhydrophobe, 3100; approx. wt. % of hydrophile, 40%), Pluronic® R 25R4(ave. MW: 3600; approx. MW of hydrophobe, 2500; approx. wt. % ofhydrophile, 40%), Pluronic® R 22R4 (ave. MW: 3350; approx. MW ofhydrophobe, 2200; approx. wt. % of hydrophile, 40%), Pluronic® R 17R4(ave. MW: 3650; approx. MW of hydrophobe, 1700; approx. wt. % ofhydrophile, 40%), Pluronic® R 25R5 (ave. MW: 4320; approx. MW ofhydrophobe, 2500; approx. wt. % of hydrophile, 50%), Pluronic® R 10R5(ave. MW: 1950; approx. MW of hydrophobe, 1000; approx. wt. % ofhydrophile, 50%), Pluronic® R 25R8 (ave. MW: 8550; approx. MW ofhydrophobe, 2500; approx. wt. % of hydrophile, 80%), Pluronic® R 17R8(ave. MW: 7000; approx. MW of hydrophobe, 1700; approx. wt. % ofhydrophile, 80%), and Pluronic® R 10R8. (ave. MW: 4550; approx. MW ofhydrophobe, 1000; approx. wt. % of hydrophile, 80%).

Other commercially available poloxamers which may be screened for theirability to enhance the immune response according to the presentinvention include compounds that are block copolymer of polyethylene andpolypropylene glycol such as Synperonic® L121, Synperonic® L122,Synperonic® P104, Synperonic® P105, Synperonic® P123, Synperonic® P85and Synperonic® P94; and compounds that are nonylphenyl polyethyleneglycol such as Synperonic® NP10, Synperonic® NP30 and Synperonic® NP5.

Other poloxamers which may be screened for their ability to enhance theimmune response according to the present invention include a polyetherblock copolymer comprising an A-type segment and a B-type segment,wherein the A-type segment comprises a linear polymeric segment ofrelatively hydrophilic character, the repeating units of whichcontribute an average Hansch-Leo fragmental constant of about −0.4 orless and have molecular weight contributions between about 30 and about500, wherein the B-type segment comprises a linear polymeric segment ofrelatively hydrophobic character, the repeating units of whichcontribute an average Hansch-Leo fragmental constant of about −0.4 ormore and have molecular weight contributions between about 30 and about500, wherein at least about 80% of the linkages joining the repeatingunits for each of the polymeric segments comprise an ether linkage; (b)a block copolymer having a polyether segment and a polycation segment,wherein the polyether segment comprises at least an A-type block, andthe polycation segment comprises a plurality of cationic repeatingunits; and (c) a polyether-polycation copolymer comprising a polymer, apolyether segment and a polycationic segment comprising a plurality ofcationic repeating units of formula —NH—R⁰, wherein R⁰ is a straightchain aliphatic group of 2 to 6 carbon atoms, which may be substituted,wherein said polyether segments comprise at least one of an A-type ofB-type segment. See U.S. Pat. No. 5,656,611, by Kabonov, et al., whichis incorporated herein by reference in its entirety.

Other auxiliary agents which may be screened for their ability toenhance the immune response according to the present invention include,but are not limited to Acacia (gum arabic); the poloxyethylene etherR—O—(C₂H₄O)_(x)—H (BRIJ®), e.g., polyethylene glycol dodecyl ether(BRIJ® 35, x=23), polyethylene glycol dodecyl ether (BRIJ® 30, x=4),polyethylene glycol hexadecyl ether (BRIJ® 52 x=2), polyethylene glycolhexadecyl ether (BRIJ® 56, x=10), polyethylene glycol hexadecyl ether(BRIJ® 58P, x=20), polyethylene glycol octadecyl ether (BRIJ® 72, x=2),polyethylene glycol octadecyl ether (BRIJ® 76, x=10), polyethyleneglycol octadecyl ether (BRIJ® 78P, x=20), polyethylene glycol oleylether (BRIJ® 92V, x=2), and polyoxyl 10 oleyl ether (BRIJ® 97, x=10);poly-D-glucosamine (chitosan); chlorbutanol; cholesterol;diethanolamine; digitonin; dimethylsulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA); glyceryl monosterate; lanolin alcohols; mono-and di-glycerides; monoethanolamine; nonylphenol polyoxyethylene ether(NP-40®); octylphenoxypolyethoxyethanol (NONIDET NP-40 from Amresco);ethyl phenol poly(ethylene glycol ether)^(n), n=11 (Nonidet® P40 fromRoche); octyl phenol ethylene oxide condensate with about 9 ethyleneoxide units (nonidet P40); IGEPAL CA 630® ((octylphenoxy)polyethoxyethanol; structurally same as NONIDET NP-40); oleicacid; oleyl alcohol; polyethylene glycol 8000; polyoxyl 20 cetostearylether; polyoxyl 35 castor oil; polyoxyl 40 hydrogenated castor oil;polyoxyl 40 stearate; polyoxyethylene sorbitan monolaurate (polysorbate20, or TWEEN-20®; polyoxyethylene sorbitan monooleate (polysorbate 80,or TWEEN-80®); propylene glycol diacetate; propylene glycol monstearate;protamine sulfate; proteolytic enzymes; sodium dodecyl sulfate (SDS);sodium monolaurate; sodium stearate; sorbitan derivatives (SPAN®), e.g.,sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60),sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), andsorbitan trioleate (SPAN® 85);2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa-hexaene (squalene);stachyose; stearic acid; sucrose; surfactin (lipopeptide antibiotic fromBacillus subtilis); dodecylpoly(ethyleneglycolether)₉ (Thesit®) MW582.9; octyl phenol ethylene oxide condensate with about 9-10 ethyleneoxide units (Triton X-100™); octyl phenol ethylene oxide condensate withabout 7-8 ethylene oxide units (Triton X-114™);tris(2-hydroxyethyl)amine (trolamine); and emulsifying wax.

In certain adjuvant compositions, the adjuvants are cytokines. Acomposition of the present invention can comprise one or more cytokines,chemokines, or compounds that induce the production of cytokines andchemokines, or a polynucleotide encoding one or more cytokines,chemokines, or compounds that induce the production of cytokines andchemokines. Examples include, but are not limited to granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), colony stimulating factor (CSF), erythropoietin (EPO),interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4),interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7),interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12),interleukin 15 (IL-15), interleukin 18 (IL-18), interferon alpha (IFNα),interferon beta (IFNβ), interferon gamma (IFNγ), interferon omega(IFNω), interferon tau (IFNτ), interferon gamma inducing factor I(IGIF), transforming growth factor beta (TGF-β), RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), Leishmaniaelongation initiating factor (LEIF), and Flt-3 ligand.

In certain compositions of the present invention, the polynucleotideconstruct may be complexed with an adjuvant composition comprising(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE). The composition may also comprise one or moreco-lipids, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE). An adjuvantcomposition comprising; GAP-DMORIE and DPyPE at a 1:1 molar ratio isreferred to herein as Vaxfectin™. See, e.g., PCT Publication No. WO00/57917, which is incorporated herein by reference in its entirety.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedprotection. For example, an increase in humoral immunity is typicallymanifested by a significant increase in the titer of antibodies raisedto the antigen, and an increase in T-cell activity is typicallymanifested in increased cell proliferation, increased cytokineproduction and/or antigen specific cytolytic activity. An adjuvant mayalso alter an immune response, for example, by changing a Th₂ responseinto a Th₁ response.

Nucleic acid molecules and/or polynucleotides of the present invention,e.g., pDNA, mRNA, linear DNA or oligonucleotides, may be solubilized inany of various buffers. Suitable buffers include, for example, phosphatebuffered saline (PBS), normal saline, Tris buffer, and sodium phosphate(e.g., 150 mM sodium phosphate). Insoluble polynucleotides may besolubilized in a weak acid or weak base, and then diluted to the desiredvolume with a buffer. The pH of the buffer may be adjusted asappropriate. In addition, a pharmaceutically acceptable additive can beused to provide an appropriate osmolarity. Such additives are within thepurview of one skilled in the art. For aqueous compositions used invivo, sterile pyrogen-free water can be used. Such formulations willcontain an effective amount of a polynucleotide together with a suitableamount of an aqueous solution in order to prepare pharmaceuticallyacceptable compositions suitable for administration to a human.

Compositions of the present invention can be formulated according toknown methods. Suitable preparation methods are described, for example,in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., MackPublishing Co., Easton, Pa. (1980), and Remington's PharmaceuticalSciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton,Pa. (1995), both of which are incorporated herein by reference in theirentireties. Although the composition may be administered as an aqueoussolution, it can also be formulated as an emulsion, gel, solution,suspension, lyophilized form, or any other form known in the art. Inaddition, the composition may contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives.

The following examples are included for purposes of illustration onlyand are not intended to limit the scope of the present invention, whichis defined by the appended claims. All references cited in the Examplesare incorporated herein by reference in their entireties.

EXAMPLES Materials and Methods

The following materials and methods apply generally to all the examplesdisclosed herein. Specific materials and methods are disclosed in eachexample, as necessary.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology (including PCR), vaccinology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed.,Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I andII (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation(B. D. Hames & S. J. Higgins eds: 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); and inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989).

Gene Construction

Constructs of the present invention are constructed based on thesequence information provided herein or in the art utilizing standardmolecular biology techniques, including, but not limited to thefollowing. First, a series complementary oligonucleotide pairs of 80-90nucleotides each in length and spanning the length of the construct aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends. Thesingle-stranded ends of each pair of oligonucleotides are designed toanneal with a single-stranded end of an adjacent oligonucleotide duplex.Several adjacent oligonucleotide pairs prepared in this manner areallowed to anneal, and approximately five to six adjacentoligonucleotide duplex fragments are then allowed to anneal together viathe cohesive single stranded ends. This series of annealedoligonucleotide duplex fragments is then ligated together and clonedinto a suitable plasmid, such as the TOPO® vector available fromInvitrogen Corporation, Carlsbad, Calif. The construct is then sequencedby standard methods. Constructs prepared in this manner, comprising 5 to6 adjacent 80 to 90 base pair fragments ligated together, i.e.,fragments of about 500 base pairs, are prepared, such that the entiredesired sequence of the construct is represented in a series of plasmidconstructs. The inserts of these plasmids are then cut with appropriaterestriction enzymes and ligated together to form the final construct.The final construct is then cloned into a standard bacterial cloningvector, and sequenced. Alternatively, wild sequences can be cloneddirectly from HCMV-infected cells (e.g. MRC-5 cells, ATCC Accession No.CCL-171, available from the American Type Culture Collection, Manassas,Va.) using PCR primers that amplify the gene of interest. Theoligonucleotides and primers referred to herein can easily be designedby a person of skill in the art based on the sequence informationprovided herein and in the art, and such can be synthesized by any of anumber of commercial nucleotide providers, for example Retrogen, SanDiego, Calif., and GENEART, Regensburg, Germany.

Plasmid Vector

Constructs of the present invention were inserted into eukaryoticexpression vector V10551. This vector is built on a modified pUC18background (see Yanisch-Perron, C., et al. Gene 33:103-119 (1985)), andcontains a kanamycin resistance gene, the human cytomegalovirusimmediate early 1 promoter/enhancer and intron A, and the bovine growthhormone transcription termination signal, and a polylinker for insertingforeign genes. See Hartikka, J., et al., Hum. Gene Ther. 7:1205-1217(1996). However, other standard commercially available eukaryoticexpression vectors may be used in the present invention, including, butnot limited to: plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS,pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6N5-His, pVAX1, and pZeoSV2(available from Invitrogen, San Diego, Calif.), and plasmid pCI(available from Promega, Madison, Wis.).

Plasmid DNA Purification

Plasmid DNA was transformed into Escherichia coli DH5α competent cellsand highly purified covalently closed circular plasmid DNA was isolatedby a modified lysis procedure (Horn, N. A., et al., Hum. Gene Ther.6:565-573 (1995)) followed by standard double CsCl-ethidium bromidegradient ultracentrifugation (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y. (1989)). Alternatively, plasmid DNAs are purified usingGiga columns from Qiagen (Valencia, Calif.) according to the kitinstructions. All plasmid preparations were free of detectablechromosomal DNA, RNA and protein impurities based on gel analysis andthe bicinchoninic protein assay (Pierce Chem. Co., Rockford Ill.).Endotoxin levels were measured using Limulus Amebocyte Lysate assay(LAL, Associates of Cape Cod, Falmouth, Mass.) and were less than 0.6Endotoxin Units/mg of plasmid DNA. The spectrophotometric A₂₆₀/A₂₈₀ratios of the DNA solutions were typically above 1.8. Plasmids wereethanol precipitated and resuspended in an appropriate solution, e.g.,150 mM sodium phosphate (for other appropriate excipients and auxiliaryagents, see U.S. Patent Application Publication 20020019358, publishedFeb. 14, 2002). DNA was stored at −20° C. until use. DNA was diluted bymixing it with 300 mM salt solutions and by adding appropriate amount ofUSP water to obtain 1 mg/ml plasmid DNA in the desired salt at thedesired molar concentration.

Plasmid Expression in Mammalian Cell Lines

The expression plasmids were analyzed in vitro by transfecting theplasmids into a well characterized mouse melanoma cell line (VM-92, alsoknown as UM-449) available from the American Type Culture Collection,Manassas, Va. Other well-characterized human cell lines may also beused, e.g. MRC-5 cells, ATCC Accession No. CCL-171. The transfection wasperformed using cationic lipid-based transfection procedures well knownto those of skill in the art. Other transfection procedures are wellknown in the art and may be used, for example electroporation andcalcium chloride-mediated transfection (Graham F. L. and A. J. van derEb Virology 52:456-67 (1973)). Following transfection, cell lysates andculture supernatants of transfected cells were evaluated to comparerelative levels of expression of HCMV antigen proteins. The samples wereassayed by western blots and ELISAs, using commercially availableanti-pp65 and anti-gB monoclonal antibodies (available, e.g., fromResearch Diagnostics Inc., Flanders N.J.), so as to compare both thequality and the quantity of expressed antigen. Additionally, in vitrotransfection assays were used to determine the effect of mixing thevarious plasmids comprising codon-optimized coding regions encoding HCMVpp65 and gB on levels of expression in human cells.

Expression products derived from human cells transfected with thevarious polynucleotide constructs were examined for molecular weight,and immunoreactive antigens (i.e., to react with HCMV antisera). Inaddition, a comparison of expression levels (both intra- andextra-cellular) of each class of expression plasmid (e.g., wild-type vs.human codon-optimized; truncated vs. full-length) was made.

Injections of Plasmid DNA

The quadriceps muscles of restrained awake mice (e.g., female 6-12 weekold BALB/c mice from Harlan Sprague Dawley, Indianapolis, Ind.) areinjected using a disposable sterile, plastic insulin syringe and 28 G ½needle (Becton-Dickinson, Franklin Likes, N.J., Cat. No. 329430) fittedwith a plastic collar cut from a micropipette tip, all as previouslydescribed (Hartikka, J., et al., Hum. Gene Ther. 7:1205-1217 (1996)).The mice are injected bilaterally in the rectus femoris muscle with 25μg of plasmid DNA (50 μg total per mouse) formulated in a salt solution(e.g. 150 mM Sodium Phosphate or phosphate buffered saline (PBS)) orwith a lipid-based delivery system.

Animal care throughout the study is in compliance with the “Guide forthe Use and Care of Laboratory Animals,” Institute of Laboratory AnimalResources, Commission on Life Sciences, National Research Council,National Academy Press, Washington, D.C., 1996 as well as with Vical'sInstitutional Animal Care and Use Committee.

Immune Correlates

Although HCMV can only infect human cells, a number of reliable animalmodels for HCMV infection are known in the art, as reviewed by Staczek,and may be used with the methods of the present invention, e.g. to testimmunogenicity or expression (Staczek, J. Microbiol Rev 54:247-65(1990)). For example, the transgenic human leukocyte antigen (HLA)A*0201.Kb mouse model may be used (Gallez-Hawkins, G. et al. Scand JImmunol 55:592-8 (2002)). A mouse model of vertical HCMV transmission isdescribed in Tang, et al., (Tang, J L, et al. Arch Virol/47:1189-95(2002)). Several models infecting human tissue implanted ontoimmunodeficient SCID or nude mice have been described (Bidanset, D J, etal., J Infect Dis 184:192-5 (2001); Pari, G S, et al, J Infect Dis177:523-8 (1998); Mocarski, E S, et al. Proc Natl Acad Sci USA 90:104-8(1993)). Athymic rats have been used to model cytomegalovirus retinitisusing HCMV (Laycock, K A, et al. Am J Ophthalmol 124:181-9 (1997)).Additionally, animal models using animal cytomegaloviruses to mimic HCMVinfection have been described, including primate models in which rhesusmacaques are infected with rhesus cytomegalovirus, and murine modelsinfected with murine cytomegalovirus (Sequar, G. et al. J Viral76:7661-71 (2002); Lockridge, K M, et al. J Viral 73:9576-83 (1999);Minamishima, Y, et al., Microbial Immunol 22:693-700 (1978)).

Example 1 Construction of an Isolated Polynucleotide Comprising aMinimally Human Codon-Optimized pp65 Coding Region, Encoding HumanCytomegalovirus pp65 with Kinase Site Deleted

VCL-6368 encodes an optimized and mutated form of the human CMV antigenpp65 cloned into the expression vector VR10551 described supra. Thisplasmid encodes a 557 amino acid protein (SEQ ID NO:6) in which aminoacids Arg435-Lys438 of the human CMV pp65 antigen have been deleted. Thecoding sequence was minimally optimized for expression in humans bychanging five codons that are rarely used in humans to correspondingcodons that are used more frequently. The five codons and changes are:Ala GCG to GCC, Arg CGT to CGC, Pro CCG to CCC, and CCA, Ser TCG to TCC,and Thr ACG to ACC. The optimized sequence is SEQ ID NO:5.

The pp65delArg435-Lys438 insert of VCL-6368 was constructed in two stepsby PCR amplification of an optimized hCMV pp65 plasmid synthesized atRetrogen Inc. (San Diego). The TOPO-hCMV-opti-pp65 plasmid (Retrogenproduct #8041-8081-4) was amplified with Expand DNA polymerase(Boehringer Mannheim) using the primer set T7 (Invitrogen Cat. #N650-02)(SEQ ID NO:21) and 65-delta-rev (SEQ ID NO:22) and the resulting productwas gel purified as a 1330 bp fragment. An overlapping 400 bp fragmentwas amplified from the same parent TOPO plasmid using the primer setM13rev (Invitrogen Cat. #18430017) (SEQ ID NO:23) and 65-delta-for (SEQID NO:24) and the product was gel purified. Ten microliters of each ofthe two PCR fragments were combined in a second PCR reaction andamplified with the T7 (SEQ ID NO:21) and M13rev primer (SEQ ID NO:23)and the 1704 bp fragment was gel purified. This fragment was cut withthe restriction enzymes Avr II and Nhe I and ligated with similarlydigested plasmid backbone DNA. The ligation mix was transformed into E.coli (XL-2 from Stratagene, Inc.) and screened by PCR for recombinantclones using the primers VR10551FOR (SEQ ID NO:25) and hCMVpp65-R (SEQID NO:26). Several PCR positive clones were picked and sequenced. Aminimally human codon-optimized clone encoding the correct Arg435-Lys438deletion form of the human CMV pp65 antigen was selected and used forfurther analysis.

Expression of VR6368 was shown by transfection of VM92 cells and Westernblot analysis using a monoclonal anti-pp65 antibody (ViroGen, lot#hCMV-pp65-4). The predicted sized protein was detected in thesupernatant and cell lysate. Even though this construct encodes anintracellular protein, a significant amount ends up in the supernatant.This is not a unique or particularly unusual phenomenon.

Example 2 Construction of an Isolated Polynucleotide Comprising aMinimally Human Codon-Optimized Glycoprotein B Coding Region, Encodingthe Secreted Human Cytomegalovirus Glycoprotein B

VCL-6365 encodes a secreted form of the human CMV antigen gB cloned intothe expression vector VR10551 described supra. This plasmid encodesamino acids 1-713 of the human CMV gB antigen (SEQ ID NO:14).Nucleotides 1-2139 of the wild-type gB coding sequence (SEQ ID NO:11)were minimally optimized for expression in humans by changing fivecodons that are rarely used in humans to five corresponding codons thatare used more frequently. The five codons and changes are: Ala GCG toGCC, Arg CGT to CGC, Pro CCG to CCC, CCT, and CCA, Ser TCG to TCC, andThr ACG to ACC. The optimized sequence is SEQ ID NO:13.

VR6365 was constructed by inserting a 2160 bp synthesized fragmentencoding amino acids 1-713 of the human CMV gB antigen inserted into theexpression vector VR-10551. Specifically, VR-10551 was digested with therestriction enzymes Nhe I and Avr II, and the 4.5 kb linear vector wasgel purified. The gB insert was obtained by digesting the minimallyhuman codon-optimized coding region encoding the secreted gB fragmentsynthesized by Retrogen Inc. (San Diego, product #7981-8031(2)-1) withthe restriction enzymes Nhe I and Avr II, then gel purifying theresulting 2160 bp fragment. The vector and insert fragments were ligatedtogether, transformed into E. coli (XL-2 from Stratagene, Inc.) andscreened by PCR for recombinant clones using the primers 10551F (SEQ IDNO:25) and hCMVgB-R (SEQ ID NO:27). Several PCR positive clones weresequenced. A clone with the correct nucleotide sequence and was giventhe designation VR6365. This clone encodes a secreted form of the humanCMV antigen gB cloned into the Nhe I-Avr II sites of the expressionvector VR10551.

Purified plasmid DNA was used to transfect the murine cell line VM92 todetermine secretion of the minimally human-codon-optimized gB.

Secretion of the minimally human-codon-optimized gB was confirmed withan ELISA assay using plates coated with supernatants from thetransfected VM92 cells. Expression and secretion was visualized withpolyclonal anti-gB serum and a commercially available anti-gB monoclonalantibody (available from Research Diagnostics Inc., Flanders, N.J.).

Example 3 Construction of an Isolated Polynucleotide Comprising a HumanCodon-Optimized CMV IE1 Coding Region, Encoding Human CytomegalovirusIE1

Plasmid VCL-6520 comprises a 1236 base-pair human codon-optimizedsynthetic DNA construct encoding exons 2 and 4 of the human CMV IE1gene, inserted into the expression vector VR-10551. The wild typesequence for exons 2 and 4 of the human CMV IE1 gene follows (SEQ ID NO:50):

GAATTCGCCGCCACCATGGAGTCCTCTGCCAAGAGAAAGATGGACCCTGATAATCCTGACGAGGGCCCTTCCTCCAAGGTGCCACGGGTCAAACAGATTAAGGTTCGAGTGGACATGGTGCGGCATAGAATCAAGGAGCACATGCTGAAAAAATATACCCAGACGGAAGAGAAATTCACTGGCGCCTTTAATATGATGGGAGGATGTTTGCAGAATGCCTTAGATATCTTAGATAAGGTTCATGAGCCTTTCGAGGAGATGAAGTGTATTGGGCTAACTATGCAGAGCATGTATGAGAACTACATTGTACCTGAGGATAAGCGGGAGATGTGGATGGCTTGTATTGATGAACTTAGGAGAAAGATGATGTATATGTGCTACAGGAATATAGAGTTCTTTACCAAGAACTCAGCCTTCCCTAAGACCACCAATGGCTGCAGTCAGGCCATGGCGGCACTGCAGAACTTGCCTCAGTGCTCCCCTGATGAGATTATGGCTTATGCCCAGAAAATATTTAAGATTTTGGATGAGGAGAGAGACAAGGTGCTCACGCACATTGATCACATATTTATGGATATCCTCACTACATGTGTGGAAACAATGTGTAATGAGTACAAGGTCACTAGTGACGCTTGTATGATGACCATGTACGGGGGCATCTCTCTCTTAAGTGAGTTCTGTCGGGTGCTGTGCTGCTATGTCTTAGAGGAGACTAGTGTGATGCTGGCCAAGCGGCCTCTGATAACCAAGCCTGAGGTTATCAGTGTAATGAAGCGCCGCATTGAGGAGATCTGCATGAAGGTCTTTGCCCAGTACATTCTGGGGGCCGATCCTCTGAGAGTCTGCTCTCCTAGTGTGGATGACCTACGGGCCATCGCCGAGGAGTCAGATGAGGAAGAGGCTATTGTAGCCTACACTTTGGCCACCGCTGGTGTCAGCTCCTCTGATTCTCTGGTGTCACCCCCAGAGTCCCCTGTACCCGCGACTATCCCTCTGTCCTCAGTAATTGTGGCTGAGAACAGTGATCAGGAAGAAAGTGAGCAGAGTGATGAGGAAGAGGAGGAGGGTGCTCAGGAGGAGCGGGAGGACACTGTGTCTGTCAAGTCTGAGCCAGTGTCTGAGATAGAGGAAGTTGCCCCAGAGGAAGAGGAGGATGGTGCTGAGGAACCCACCGCCTCTGGAGGCAAGAGCACCCACCCTATGGTGACTAGAAGCAAGGCTGACCAGTGAGGATCC

The insert in the VCL-6250 construct was synthesized by GENEARTwww.geneart.de/, Regensburg, Germany). VCL-6250 has the followingsequence (SEQ ID NO:28):

10ATATCGCCGCCACCATGGAGTCTAGCGCCAAGAGGAAGATGGACCCCGACAACCCTGATGAGGGCCCTAGCAGCAAGGTGCCCCGGGTGAAGCAGATCAAGGTGCGGGTGGACATGGTGCGGCACAGGATCAAGGAACACATGCTGAAGAAGTACACCCAGACCGAGGAGAAGTTCACCGGCGCCTTCAATATGATGGGCGGCTGCCTGCAGAATGCCCTGGACATCCTGGACAAGGTGCACGAGCCCTTCGAGGAGATGAAGTGCATCGGCCTGACCATGCAGAGCATGTACGAGAACTACATCGTGCCCGAGGACAAGAGGGAGATGTGGATGGCCTGCATCGACGAGCTGCGGCGGAAGATGATGTACATGTGCTACCGGAACATCGAGTTCTTCACCAAGAACAGCGCCTTCCCCAAGACCACCAACGGATGCTCTCAGGCCATGGCCGCCCTGCAGAATCTGCCTCAGTGCAGCCCCGATGAGATCATGGCCTACGCCCAGAAGATCTTCAAGATCCTGGACGAGGAGAGGGATAAGGTGCTGACCCACATCGACCACATCTTCATGGACATCCTGACCACCTGCGTGGAGACCATGTGCAACGAGTACAAGGTGACCAGCGACGCCTGCATGATGACAATGTACGGCGGCATCAGCCTGCTGAGCGAGTTCTGCAGAGTGCTGTGCTGCTACGTGCTGGAGGAGACCTCTGTGATGCTGGCCAAGAGGCCCCTGATCACCAAGCCTGAGGTGATCAGCGTGATGAAGCGGCGGATCGAGGAGATCTGCATGAAGGTGTTCGCCCAGTACATCCTGGGAGCCGACCCTCTGAGAGTGTGTAGCCCCAGCGTGGATGACCTGAGAGCCATCGCCGAGGAATCTGATGAAGAGGAGGCCATCGTGGCCTATACACTGGCCACAGCCGGCGTGTCTAGCAGCGATAGCCTGGTGAGCCCTCCTGAGTCTCCTGTGCCTGCCACAATCCCTCTGAGCAGCGTGATCGTGGCCGAGAACAGCGATCAGGAGGAGAGCGAGCAGTCTGATGAGGAAGAGGAAGAGGGAGCCCAGGAGGAGAGAGAGGATACCGTGAGCGTGAAGAGCGAGCCTGTGAGCGAGATCGAAGAGGTGGCCCCTGAGGAAGAAGAGGATGGCGCCGAGGAGCCTACAGCCAGCGGCGGCAAGTCAACACACCCCATGGTGACCAGAAGCAAGGCCGACCAGTAAGGATCC

VCL-6250 was constructed by isolating the EcoR5-BamHI IE1 syntheticinsert and ligating it into the expression vector VR-10551, describedabove. Specifically, VR-10551 was digested with restriction enzymes andgel purified, as described in the preceding examples. The vector andinsert fragments were ligated together, transformed into E. coli DH10Bcells (available, e.g., from Invitrogen). Selected recombinant plasmidswere completely sequences using the primers synthesized according to thefollowing table:

TABLE 9 Primers Primer Sequence SEQ ID NO 2944SCTG CGC CTT ATC CGG TAA CT SEQ ID NO: 33 5876 CAG TGA GGC ACC TAT CTC AGSEQ ID NO: 34 5760 CAC CAT GAG TGA CGA CTG AA SEQ ID NO: 35 5761TTA ATC GCG GCC TCG AGC AA SEQ ID NO: 36 5762 GGC TCA TGT CCA ACA TTA CCSEQ ID NO: 37 931S GAG ACG CCA TCC ACG CTG TT SEQ ID NO: 38 5874CAG ACT TAG GCA CAG CAC AA SEQ ID NO: 39 5104 GAG CGA GGA AGC GGA AGA GTSEQ ID NO: 40 3054A CCG CCT ACA TAC CTC GCT CT SEQ ID NO: 41 5767GAG CAT TAC GCT GAC TTG AC SEQ ID NO: 42 5768 ATG CCT CTT CCG ACC ATC AASEQ ID NO: 43 5770 GGC GGT AAT GTT GGA CAT GA SEQ ID NO: 44 847AGGC GGA GTT GTT ACG ACA TT SEQ ID NO: 45 5772 CAT TGT GCT GTG CCT AAG TCSEQ ID NO: 46 GA seqF1 CCA GAC CGA GGA GAA GTT CA SEQ ID NO: 47 GA seqF2TGC TGG AGG AGA CCT CTG TG SEQ ID NO: 48 GA seqR2TCG ATC CGC CGC TFC ATC AC SEQ ID NO: 49

Purified VCL-6250 DNA was used to transfect the murine cell line VM92 todetermine expression of the IE1 protein. Expression of IE1 was confirmedwith a Western Blot assay. Expression was visualized with a commerciallyavailable anti-IE1 monoclonal antibody (available from ChemiconInternational, Temecula, Calif.).

Example 4 Preparation of Vaccine Formulations

In each of the following methods, HCMV antigen-encoding plasmids of thepresent invention are formulated with the poloxamer system, describedherein as VF-P1205-02A. VF-P1205-02A refers to a poloxamer-baseddelivery system consisting of the non-ionic block copolymer, CRL 1005,and a cationic surfactant, BAK (Benzalkonium chloride 50% solution,available from Ruger Chemical Co. Inc.). Specific final concentrationsof each component of the formulae are described in the followingmethods, but for any of these methods, the concentrations of eachcomponent may be varied by basic stoichiometric calculations known bythose of ordinary skill in the art to make a final solution having thedesired concentrations.

For example, the concentration of CRL 1005 is adjusted depending on, forexample, transfection efficiency, expression efficiency, orimmunogenicity, to achieve a final concentration of between about 1mg/ml to about 75 mg/ml, for example, about 1 mg/ml, about 2 mg/ml,about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6.5 mg/ml, about 7mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml,about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml,about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, or about 75 mg/ml of CRL1005.

Similarly the concentration of DNA is adjusted depending on manyfactors, including the amount of a formulation to be delivered, the ageand weight of the subject, the delivery method and route and theimmunogenicity of the antigen being delivered. In general, formulationsof the present invention are adjusted have a final concentration fromabout 1 ng/ml to about 30 mg/ml of plasmid (or other polynucleotide).For example, a formulation of the present invention may have a finalconcentration of about 1 ng/ml, about 5 ng/ml, about 10 ng/ml, about 50ng/ml, about 100 ng/ml, about 500 ng/ml, about 1 μg/ml, about 5 μg/ml,about 10 μg/ml, about 50 μg/ml, about 200 μg/ml, about 400 μg/ml, about600 μg/ml, about 800 μg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5,about 3 mg/ml, about 3.5, about 4 mg/ml, about 4.5, about 5 mg/ml, about5.5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml,about 10 mg/ml, about 20 mg/ml, or about 30 mg mg/ml of a plasmid.

Certain formulations of the present invention include a cocktail ofplasmids, for example, a mixture of two or more of plasmids VCL-6365,VCL-6368, or VCL-6520 of the present invention, and optionally plasmidscomprising codon-optimized or non-codon-optimized coding regionsencoding other HCMV antigens, e.g., an antigenic portion if HCMV IE1,and/or plasmids encoding immunity enhancing proteins, e.g., cytokines.Various plasmids desired in a cocktail are combined together in PBS orother diluent prior to the addition to the other ingredients.Furthermore, plasmids may be present in a cocktail at equal proportions,or the ratios may be adjusted based on, for example, relative expressionlevels of the antigens or the relative immunogenicity of the encodedantigens. Thus, various plasmids in the cocktail may be present in equalproportion, or up to twice or three times, or more, as much of oneplasmid may be included relative to other plasmids in the cocktail.

Additionally, the concentration of BAK may be adjusted depending on, forexample, a desired particle size and improved stability: Indeed, incertain embodiments, formulations of the present invention include CRL1005 and DNA, but are free of BAK. In general BAK-containingformulations of the present invention are adjusted to have a finalconcentration of BAK from about 0.05 mM to about 0.5 mM. For example, aformulation of the present invention may have a final BAK concentrationof about 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.

The total volume of the formulations produced by the methods below maybe scaled up or down, by choosing apparatus of proportional size.Finally, in carrying out any of the methods described below, the threecomponents of the formulation, BAK, CRL 1005, and plasmid DNA, may beadded in any order. In each of these methods described below the term“cloud point” refers to the point in a temperature shift, or othertitration, at which a clear solution becomes cloudy, i.e., when acomponent dissolved in a solution begins to precipitate out of solution.

A. Thermal Cycling of a Pre-Mixed Formulation

This example describes the preparation of a formulation comprising 0.3mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a total volume of 3.6ml. The ingredients are combined together at a temperature below thecloud point and then the formulation is thermally cycled to roomtemperature (above the cloud point) several times, according to theprotocol outlined in FIG. 8.

A 1.28 mM solution of BAK is prepared in PBS, 846 μl of the solution isplaced into a 15 ml round bottom flask fitted with a magnetic stirringbar, and the solution is stirred with moderate speed, in an ice bath ontop of a stirrer/hotplate (hotplate off) for 10 minutes. CRL 1005 (27μl) is then added using a 100 μl positive displacement pipette and thesolution is stirred for a further 60 minutes on ice. Plasmids VCL-6365and VCL-6368, and optionally, additional plasmids encoding, e.g.,additional HCMV antigens, e.g., VLC-6520, are mixed together at desiredproportions in PBS. In the present example, 2.73 ml of a solutioncontaining 3.2 mg/ml VCL-6365 and 3.2 mg/ml VCL-6368 (6.4 mg/ml totalDNA) is added drop wise, slowly, to the stirring solution over 1 minusing a 5 ml pipette. The solution at this point (on ice) is clear sinceit is below the cloud point of the poloxamer and is further stirred onice for 15 min. The ice bath is then removed, and the solution isstirred at ambient temperature for 15 minutes to produce a cloudysolution as the poloxamer passes through the cloud point.

The flask is then placed back into the ice bath and stirred for afurther 15 minutes to produce a clear solution as the mixture is cooledbelow the poloxamer cloud point. The ice bath is again removed and thesolution stirred at ambient temperature for a further 15 minutes.Stirring for 15 minutes above and below the cloud point (total of 30minutes), is defined as one thermal cycle. The mixture is cycled sixmore times. The resulting formulation may be used immediately, or may beplaced in a glass vial, cooled below the cloud point, and frozen at −80°C. for use at a later time.

B. Thermal Cycling, Dilution and Filtration of a Pre-Mixed Formulation,Using Increased Concentrations of CRL 1005

This example describes the preparation of a formulation comprising 0.3mM BAK, 34 mg/ml or 50 mg/ml CRL 1005, and 2.5 mg/ml of DNA in a finalvolume of 4.0 ml. The ingredients are combined together at a temperaturebelow the cloud point, then the formulation is thermally cycled to roomtemperature (above the cloud point) several times, diluted, and filteredaccording to the protocol outlined in FIG. 9.

Plasmids VCL-6365 and VCL-6368, and optionally, additional plasmidsencoding, e.g., additional HCMV antigens, e.g., VLC-6520, are mixedtogether at desired proportions in PBS. For the formulation containing34 mg/ml CRL 1005, 1.55 ml of a solution containing about 3.2 mg/mlVCL-6365 and about 3.2 mg/ml VCL-6368 (about 6.4 mg/ml total DNA) isplaced into the 15 ml round bottom flask fitted with a magnetic stirringbar, and for the formulation containing 50 mg/ml CRL 1005, 1.52 ml of asolution containing about 3.2 mg/ml VCL-6365 and about 3.2 mg/mlVCL-6368 (about 6.4 mg/ml total DNA) is placed into the 15 ml roundbottom flask fitted with a magnetic stirring bar, and the solutions arestirred with moderate speed, in an ice bath on top of a stirrer/hotplate(hotplate off) for 10 minutes. CRL 1005 (68 μl for 34 mg/ml finalconcentration, and 100 μl for 50 mg/ml final concentration) is thenadded using a 100 μl positive displacement pipette and the solution isstirred for a further 30 minutes on ice. A 1.6 mM solution of BAK isprepared in PBS, and 375 μl is then added drop wise, slowly, to thestirring 34 mg/ml or 50 mg/ml mixtures, over 1 min using a 1 ml pipette.The solutions at this point are clear since they are below the cloudpoint of the poloxamer and are stirred on ice for 30 min. The ice bathsare then removed; the solutions stirred at ambient temperature for 15minutes to produce cloudy solutions as the poloxamer passes through thecloud point.

The flasks are then placed back into the ice baths and stirred for afurther 15 minutes to produce clear solutions as the mixtures cooledbelow the poloxamer cloud point. The ice baths are again removed and thesolutions stirred for a further 15 minutes. Stirring for 15 minutesabove and below the cloud point (total of 30 minutes), is defined as onethermal cycle. The mixtures are cycled two more times.

In the meantime, two Steriflip® 50 ml disposable vacuum filtrationdevices, each with a 0.22 μm Millipore Express® membrane (available fromMillipore, cat #SCGP00525) are placed in an ice bucket, with a vacuumline attached and left for 1 hour to allow the devices to equilibrate tothe temperature of the ice. The poloxamer formulations are then dilutedto 2.5 mg/ml DNA with PBS and filtered under vacuum.

The resulting formulations may be used immediately, or may betransferred to glass vials, cooled below the cloud point, and frozen at−80° C. for use at a later time.

C. A Simplified Method without Thermal Cycling

This example describes a simplified preparation of a formulationcomprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a totalvolume of 3.6 ml. The ingredients are combined together at a temperaturebelow the cloud point and then the formulation is simply filtered andthen used or stored, according to the protocol outlined in FIG. 10.

A 0.77 mM solution of BAK is prepared in PBS, and 780 μl of the solutionis placed into a 15 ml round bottom flask fitted with a magneticstirring bar, and the solution is stirred with moderate speed, in an icebath on top of a stirrer/hotplate (hotplate off) for 15 minutes. CRL1005 (15 μl) is then added using a 100 μl positive displacement pipetteand the solution is stirred for a further 60 minutes on ice. PlasmidsVCL-6365 and VCL-6368, and optionally, additional plasmids encoding,e.g., additional HCMV antigens, e.g., VLC-6250, are mixed together atdesired proportions in PBS. In the present example, about 1.2 ml of asolution containing about 4.1 mg/ml VCL-6365 and about 4.2 mg/mlVCL-6368 (about 8.3 mg/ml total DNA) is added drop wise, slowly, to thestirring solution over 1 min using a 5 ml pipette. The solution at thispoint (on ice) is clear since it is below the cloud point of thepoloxamer and is further stirred on ice for 15 min.

In the meantime, two Steriflip® 50 ml disposable vacuum filtrationdevices, each with a 0.22 μm Millipore Express® membrane (available fromMillipore, cat #SCGP00525) are placed in an ice bucket, with a vacuumline attached and left for 1 hour to allow the devices to equilibrate tothe temperature of the ice. The poloxamer formulation was then filteredunder vacuum, below the cloud point and then allowed to warm above thecloud point. The resulting formulations may be used immediately, or maybe transferred to glass vials, cooled below the cloud point and thenfrozen at −80° C. for use at a later time.

Example 5 Animal Immunization

The immunogenicity of expression products encoded by one or more of thecodon-optimized polynucleotides described in Examples 1, 2 and 3, andoptionally the codon-optimized polynucleotides described in Example 4,are evaluated based on each plasmid's ability to mount an immuneresponse in vivo. Plasmids are tested individually and in combinationsby injecting single constructs as well as multiple constructs.Immunizations are initially carried out in animals, such as mice,rabbits, goats, sheep, primates, or other suitable animal, byintramuscular (IM) injections. Serum is collected from immunizedanimals, and the immune response is quantitated. The tests ofimmunogenicity further include measuring antibody titer, neutralizingantibody titer, T cell cytokine production and T cell cytolyticactivity. Correlation to protective levels in humans are made accordingto methods well known by those of ordinary skill in the art. See “immunecorrelates,” above.

A. DNA Formulations

Plasmid DNA is formulated by any of the methods described in Example 4.Alternatively, plasmid DNA is prepared as described above and dissolvedat a concentration of about 0.1 mg/ml to about 10 mg/ml, preferablyabout 1 mg/ml, in PBS with or without transfection-facilitating cationiclipids, e.g., DMRIE/DOPE at a 4:1 DNA:lipid mass ratio. Alternative DNAformulations include 150 mM sodium phosphate instead of PBS, adjuvants,e.g., Vaxfectin™ at a 4:1 DNA: Vaxfectin™ mass ratio, mono-phosphoryllipid A (detoxified endotoxin) from S. minnesota (MPL) andtrehalosedicorynomycolateAF (TDM), in 2% oil (squalene)-Tween 80-water(MPL+TDM, available from Sigma/Aldrich, St. Louis, Mo., (catalog#M6536)), a solubilized mono-phosphoryl lipid A formulation (AF,available from Corixa), or(±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminiumchloride (compound # VC1240) (see Shriver, J. W. et al., Nature415:331-335 (2002), and P.C.T. Publication No. WO 02/00844 A2, each ofwhich is incorporated herein by reference in its entirety).

B. Animal Immunizations

Codon-optimized and wild type DNA plasmids encoding secreted gB andpp65, and their respective mutant variants, as described above, areinjected into BALB/c mice as single plasmids, as either DNA in PBS orformulated with the poloxamer-based delivery system: 3 mg/ml DNA, 34 or50 mg/ml CRL 1005, and 0.3 mM BAK. Groups of 10 mice are immunized threetimes, at biweekly intervals, and serum is obtained to determineantibody titers to each of the antigens. Groups are also included inwhich mice are immunized with a trivalent preparation, containing eachof the three plasmids in equal mass. The study design for each plasmidis shown in Table 10, and a typical immunization protocol is shown inTable 11.

TABLE 10 Study Design for Plasmids Group Number of animals DNA in PBS 10DNA formulated with CRL 1005 and BAK 10 Plasmid backbone (VR10551), DNAin PBS 5

TABLE 11 Immunization Schedule Day Immunization −3 Pre-bleed 0 Plasmidinjections, intramuscular, bilateral in rectus femoris, 25 μg/leg 14Plasmid injections, intramuscular, bilateral in rectus femoris, 25μg/leg 20 Serum collection 28 Plasmid injections, intramuscular,bilateral in rectus femoris, 25 μg/leg 35 Serum collection

Serum antibody titers are determined by ELISA with recombinant proteinsor transfection supernatants and lysates from transfected VM-92 cells orvirus-infected cell lysates.

C. Production of HCMV pp65 and gB Antisera in Animals

Plasmid DNA encoding HCMV pp65, gB, IE1 or fragments, variants orderivatives thereof is prepared according to the immunization schemedescribed above and injected into a suitable animal for generatingpolyclonal antibodies. Serum is collected and the antibody titered asabove. The titer of anti-HCMV peptide antibodies in serum from immunizedanimals may be increased by selection of anti-peptide antibodies, forinstance, by adsorption to the peptide on a solid support and elution ofthe selected antibodies according to methods well known in the art.

Monoclonal antibodies are also produced using hybridoma technology(Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. Immunol.6:511 (1976); Kohler, et al, Eur. J. Immunol. 6:292 (1976); Hammerling,et al., in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981), pp. 563-681, each of which is incorporated herein by referencein its entirety). In general, such procedures involve immunizing ananimal (preferably a mouse) as described above. Suitable cells can berecognized by their capacity to bind anti-HCMV pp65, gB antibody or IE1antibody. Such cells may be cultured in any suitable tissue culturemedium; however, it is preferable to culture cells in Earle's modifiedEagle's medium supplemented with 10% fetal bovine serum (inactivated atabout 56° C.), and supplemented with about 10 g/1 of nonessential aminoacids, about 1,000 U/ml of penicillin, and about 100 g/ml ofstreptomycin. The splenocytes of such mice are extracted and fused witha suitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2/0), availablefrom the American Type Culture Collection, Rockville, Md. After fusion,the resulting hybridoma cells are selectively maintained in HAT medium,and then cloned by limiting dilution as described by Wands et al.,Gastroenterology 80:225-232 (1981), incorporated herein by reference inits entirety. The hybridoma cells obtained through such a selection arethen assayed to identify clones which secrete antibodies capable ofbinding HCMV pp65 or gB.

Alternatively, additional antibodies capable of binding to HCMV pp65 orgB may be produced in a two-step procedure through the use ofanti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, HCMV pp65 or gB specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the HCMV protein-specific antibody can be blocked by HCMVpp65 or gB. Such antibodies comprise anti-idiotypic antibodies to theHCMV protein-specific antibody and can be used to immunize an animal toinduce formation of further HCMV pp65 or gB-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, HCMV pp65 orgB-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

It may be preferable to use “humanized” chimeric monoclonal antibodies.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric antibodies are known in the art. See, forreview, Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi, etal., EP 171496; Morrison, et al., EP 173494; Neuberger, et al., WO8601533; Robinson, et al., WO 8702671; Boulianne, et al., Nature 312:643(1984); Neuberger, et al., Nature 314:268 (1985).

These antibodies are used, for example, in diagnostic assays, as aresearch reagent, or to further immunize animals to generateHCMV-specific anti-idiotypic antibodies. Non-limiting examples of usesfor anti-HCMV antibodies include use in Western blots, ELISA(competitive, sandwich, and direct), immunofluorescence, immunoelectronmicroscopy, radioimmunoassay, immunoprecipitation, agglutinationeassays, immunodiffisuon, immunoelectrophoresis, and epitope mapping(Weir, D. Ed. Handbook of Experimental Immunology, 4^(th) ed. Vols. Iand II, Blackwell Scientific Publications (1986)).

Example 6 Quantitative, Real Time RT-PCR Analysis of mRNA Expression ofConstructs Encoding HCMV pp65 and gB, and Fragments, Variants andDerivatives Thereof

Quantitation of the mRNA levels expressed from the HCMV pp65, gB and IE1constructs is a valuable biological marker for gene activity. Variousmethods can be used to measure the levels of mRNA, such as Northernblots, slot blots, and other techniques known to those skilled in theart. However, a rapid method based on real-time RT-PCR provides anefficient, reliable means to monitor gene activity. One such system isthe TaqMan® RT-PCR assay used with an ABI PRISM® Sequence DetectionSystem, both available from Applied Biosystems, Inc. (Foster City,Calif.).

Briefly, RNA is extracted using conventional or commercially availabletechniques. After extraction, the RNA is aliquotted into opticallytransparent tubes or wells of a microtiter plate containing the providedbuffers, enzymes, and reagents supplied with the appropriate kit, e.g.,TaqMan® Gold RT-PCR Kit (Applied Biosystems, Inc., Foster City, Calif.).Additionally, the construct specific primers and probe, which can bedesigned by a person skilled in the art based on the sequences describedherein, or commercially, e.g., ABI PRISM®. Primers & TaqMan® ProbesSynthesis Service (Applied Biosystems, Inc., Foster City, Calif.) areadded. The samples are placed in the ABI PRISM® Sequence DetectionSystem, a thermocycler coupled to a laser capable of exciting thefluorophores present on the probe and a suitable detection system.Initially, the RNA is reverse transcribed into DNA, then thermostableDNA polymerase and sequence-specific primers contained in the reactionsolution initiate the temperature-controlled amplification cycles. Theprobe used for detection of the amplification product is labeled with alow energy fluorophore (the reporter) and a high energy fluorophore (thequencher), which prevents emissions of the reporter from being detectedif the quencher is closely associated with the reporter throughfluorescence resonance energy transfer (FRET). At the beginning of thereaction cycle, the probe is in excess, so the majority remainsunhybrized and intact, resulting in no signal. However, as the DNAproduct accumulates, a higher proportion of the probe is bound to theDNA. The bound probe is then degraded by the 5′ nuclease activity of theDNA polymerase used for the amplification, which releases the reporterfrom the quencher and creates a detectable signal. As the PCR reactionprogresses and the amplified product accumulates, more of the probe isdegraded, inducing a greater signal that is recorded. The number ofamplification cycles necessary to detect a signal (Ct) is directlyproportional to the amount of starting template, or construct mRNA. Bycomparing Ct values between the sample and controls starting with aknown amount of RNA, it is possible to quantitate the amount of mRNAexpressed in cells transfected with plasmids containing the HCMVconstructs. See the Applied Biosystem, Inc. tutorial “Real-Time PCR Vs.Traditional PCR” on the world wide web atwww.appliedbiosystems.com/support/tutorials/, visited Nov. 15, 2002.Other real time detection systems include “Molecular Beacon” probes,see, e.g., U.S. Pat. No. 6,103,476 to Kramer and Tyagi, which isincorporated herein by reference.

For the in vitro studies, suitable cells are seeded into 24 well tissueculture plates. Once the cells are at an appropriate cell density,plasmid DNA containing codon-optimized and non-codon-optimized HCMVconstructs or appropriate controls, e.g. negative controls containingthe plasmid backbone with no HCMV construct, is used to transfect thecells. At various time-points post-transfection, the cells are collectedfor RNA extraction, for example with 4M guanidinium thiocyanate followedby phenol extraction. Cells collected from in vivo studies are also usedfor RNA extraction. The extracted total RNA is quantitated by measuringthe absorbance of the sample at 260 nm, diluted according to the Taqman®kit instructions (Applied Biosystems, Inc., Foster City, Calif.), andaliquotted into 386 well plates suitable for real-time PCR containingthe buffers, nucleotides, and enzymes necessary. Controls containingknown amounts of starting RNA are included in the assay, and optionallyan internal standard may be included in the samples for qualityassurance. This internal standard is typically an unrelated geneproduct, usually an abundant endogenous RNA. Primers and probes specificfor the construct and optionally internal standard are also included.The primers are designed and synthesized in the same manner asconventional PCR primers, which is a routine task for one of skill inthe art. To ensure reproducibility and specificity, multiple primer setsare used in the reaction, each targeting different regions of theconstruct. The primer is synthesized in a similar manner, but thefluorophores, e.g. FAM and TAMRA, are covalently attached byconventional methods. The reaction proceeds as described above, and theresulting Ct values of the samples are compared to those of thecontrols. Starting quantities of the mRNA are interpolated using thecontrol Ct values.

After mRNA quantitation, the mRNA level is correlated with proteinexpression, both intracellular and secreted. Supernatant is collectedfrom the tissue culture medium (or from the supernatant of centrifugedcells collected in vivo) at various time-points post-transfection.Additionally, a suitable′ number of cells are retained after harvestingfor use in protein extraction. Western blots, slot blots, ELISA andother protein quantitation techniques are used to measure the HCMVprotein levels produced by the transfected cells.

Example 7 Demonstration of Immunogenicity Plasmids Encoding Human CMVAntigens General Experimental Procedure

The experimental procedure for the following example is as describedabove, with particular parameters and materials employed as describedherein.

Plasmids

As described above, constructs of the present invention were insertedinto the expression vector VR10551.

VR10551 is an expression vector without any transgene insert (backbonefor the HCMV plasmids).

VR6365 contains the coding sequence for a secreted version of human CMVgB (amino acids 1-713) cloned into the VR10551 expression vector(Example 1). The DNA was prepared using Qiagen plasmid purificationkits, and was characterized and formulated with the VF-P1205-02Apoloxamer-based delivery system.

VR6368 contains the coding sequence of the full-length HCMV pp65,deleted of residues ⁴³⁵RKRK⁴³⁸ in the putative kinase domain, clonedinto the VR10551 expression vector (Example 2). The DNA was preparedusing Qiagen plasmid purification kits, and was characterized andformulated with the VF-P1205-02A poloxamer-based delivery system, asabove.

Poloxamer Formulation

The VF-P1205-02A poloxamer-based delivery system was formulated using aprotocol equivalent to Example 4B, with an initial DNA, poloxamer andBAK concentration of 5.0 mg/ml, 7.5 mg/ml and 0.3 mM, respectively.Formulations were diluted with PBS at room temperature to the requiredexperimental concentrations prior to injection.

Vaccination Regimen

Groups of nine, 6- to 8-week old female BALB/c mice(Harlan-Sprague-Dawley) received intramuscular (rectus femoris)injections containing 100 μg of pp65 DNA, 100 μg of gB DNA, or 100 μgeach of pp65 and gB DNA delivered with PBS or the CRL 1005 poloxamerformulation described above. Control mice received 100 μg of pp65 DNA or100 μg of gB DNA mixed with 100 μg of non-coding, vector DNA (VR10551)delivered with PBS or VF-P1205-02A. All mice received two vaccinations(administered on days 0 and 13) containing a total of 200 μg of DNA, 100μg pp65 DNA and the 100 μg gB DNA. Sera were collected after the first(day 11) and second (day 22) vaccinations, and gB- and pp65-specificantibody responses were measured by ELISA and immunoblot analysis,respectively.

Recombinant gB Enzyme Linked Immunosorbent Assay (ELISA)

Sera were collected from the mice vaccinated according to the regimendescribed above. Anti-gB IgG titers were determined using a recombinantCMV gB Enzyme Linked Immunosorbent Assay (ELISA).

Ninety six-well, half area, high-binding EIA (Enzyme ImmunoAssay) plateswere coated with recombinant CMV gB at a concentration of 0.05 μg/well(50 μL/well) in Borate Buffered Saline (BBS) buffer at 4° C. overnight.Plates were covered with an adhesive plate sealer for all incubations.After coating, plates were blotted on paper towels and 100 μL ofblocking buffer (0.1% [w/v] BSA in BBS) was added to each well. Sealedplates were incubated at room temperature for 2 hours and were thenstored at 4° C. until sera had been diluted. Sera were diluted in 0.5%(w/v) BSA in BBS in Eppendorf tubes, and were mixed by inversion andbrief vortexing. Blocked plates were blotted and 100 μL of diluted serumwas added to each well. Plates were sealed and incubated overnight at 4°C. Plates were then washed on a four wash cycle on an automated platewasher with 0.1% (v/v) Tween-20 in BBS and were blotted on paper towels.Alkaline phosphate labeled anti-mouse IgG Fc secondary antibody wasdiluted 1:2000 in 0.5% (w/v) BSA in BBS and 80 μL of diluted secondaryantibody was added to each well. Plates were sealed and were incubatedat room temperature for 2 hours. Plates were washed again on the fourwash cycle on the automated plate washer and were blotted on papertowels. Fifty microliters of developing solution (1 mg/mlpara-nitrophenyl phosphate in 50 mM sodium bicarbonate buffer, pH 9.8and 1 mM MgCl₂) was added to each well, plates were sealed and incubatedat room temperature. Absorbance at 405 nm, A₄₀₅, (single wavelength) wasread on the plate reader. Titers were determined as the dilution atwhich the mean absorbance value of the immune serum was at least twicethat of the mean absorbance value for the pre-immune serum at a dilutionof 1:100.

Immunoblots to Detect pp65

Lysates from murine melanoma VM92 cells transfected with either VR6368or VR10551 were made directly in 1× NuPAGE LDS sample buffer and werestored at −80° C. until needed. After thawing at room temperature, onetenth of the sample volume of 0.5 MM dithiothreitol was added to eachsample. Samples were then heated at 85° C. for 10 min and were cooledimmediately on ice prior to loading on NuPAGE 4-12% Bis-Tris gels.Electrophoresis was carried out at 200 V for 60 minutes at roomtemperature. For transfer of proteins, polyvinylidene difluoride (PVDF)membranes were first soaked in methanol for 30 s and then equilibratedin 1× NuPAGE transfer buffer containing 20% (v/v) methanol. Proteinswere transferred from gels to PVDF membranes at 30V for 60 min at roomtemperature. After protein transfer, membranes were rinsed in milli-Qwater and then blocked for 45 min at room temperature in 1% (w/v) BSA inBBS on an orbital shaker. After blocking, membranes were stored at 4° C.in 1% (w/v) BSA in BBS for no longer than 24 hr. Blots were cut intostrips and were incubated in mouse immune serum diluted in 0.5% (w/v)BSA in BBS at room temperature overnight on an orbital shaker. Afterwashing in BBS, the strips were incubated in secondary antibody (goatanti-mouse IgG Fcγ conjugated to alkaline phosphatase) at roomtemperature for 2.5 hr. Strips were then washed again in BBS and weredeveloped in alkaline phosphatase substrate solution for 10 min at roomtemperature. Strips were then rinsed thoroughly in distilled water andwere allowed to dry at room temperature between paper towels.

Mice were vaccinated with gB plasmid (VR6365) or gB/pp65 plasmidcombination, as described above. The anti-gB IgG titers, measured aftertwo vaccinations in mice vaccinated with gB plasmid (VR6365), alone orin combination with pp65 plasmid (VR6368) are given below:

TABLE 12 Anti-gB IgG Titers Following 2^(nd) Vaccination mean reciprocaltiter geometric mean Group (range) reciprocal titer gB 42,667 34,836(poloxamer formulation) (12,800-102,400) Combination 17,244 13,825(poloxamer formulation) (1,600-25,600) gB 29,867 27,650 (naked DNA)(12,800-51,200)  Combination 10,667 8,709 (naked DNA) (3,200-25,600)

All mice vaccinated with plasmid DNA encoding HCMV gB alone or incombination, either with or without VF-P1205-02A, had detectable anti-gBIgG titers after two injections of DNA. Sera from mice injected withpp65 DNA only were pooled and tested. The binding activity for the pp65only group was the same as for the pre-bleed sera, indicating that gBspecific antibodies were not detected.

pp65 Immunoblots

Mouse sera collected after the second DNA vaccination were tested onimmunoblots of lysates from cells transfected with pp65 plasmid (VR6368)as described above to determine, qualitatively, the difference in theantibody responses to pp65 in mice vaccinated with VR6368 alone and micevaccinated with the plasmid combination. In the first set ofimmunoblots, pooled sera from each group of mice vaccinated with VR6368were tested at dilutions of 1:200, 1:400, 1:800, 1:1000 and 1:2000. Asample of pooled sera from mice vaccinated with VR6365 (gB) formulatedin VF-P1205-02A was included as a negative control. A pp65-specificmurine monoclonal antibody was included as a positive control. Eachimmunoblot strip had a lane of molecular weight standards, a lanecontaining VR6368-transfected cell lysate, and a VR10551 transfectedcell lysate control lane. All mice (nine of nine) vaccinated with pp65DNA formulated with VF-P1205-02A had detectable antibody to pp65 byimmunoblot when sera were tested at dilution of 1:200. Six of nine micevaccinated with the bivalent HCMV plasmid vaccine formulated withVF-P1205-02A had detectable antibody to pp65 by immunoblot when testedat a dilution of 1:200. Immunoblot titration of pooled sera from themice vaccinated with either the pp65 DNA formulated with VF-P1205-02A,or the bivalent HCMV plasmid vaccine formulated with VF-P1205-02A didnot reveal a marked difference in the antibody response to pp65 betweenthe groups. No pp65 antibody was detected in mice vaccinated with gB DNAalone.

Thus, plasmids VR6365 (gB) and VR6368 (pp65) elicited the production ofantigen-specific antibodies in mice that received two injections of theplasmids either alone or in combination. Although we cannot quantify theanti-pp65 antibody response using immunoblots, they do show that themajority of mice had a detectable antibody response to pp65, and thatthe combination of the two plasmids did not result in completesuppression of the response to pp65. Antibody responses to pp65 in thisstudy served as an additional readout for confirmation of production ofthis protein in vivo after vaccination with VR6368.

pp65-Specific IFN-γ ELISpot Assay

T cell responses to the DNA-encoded pp65 were determined by IFN-γELISpot assay. Splenocytes of vaccinated mice were stimulated with twoseparate pools of overlapping peptides, that, together, span the entirepp65 protein and should contain all possible T cell epitopes. Therefore,the type of the T cell (e.g., CD8⁺ or CD4⁺) that is producing IFN-γ inresponse to the peptide stimulation cannot be distinguished by thisassay method. Theoretically, these peptides can be presented in thecontext of class I or class II MHC, thus stimulating both CD8⁺ and CD4⁺T cells within the same splenocyte preparation.

In these assays the number of antigen-specific spots wereusually >10-fold more than the number in control wells. IFN-γ producingcells were detected in splenocyte preparations from VR6368-vaccinatedmice stimulated with either of the peptide pools, but approximatelythree times as many spots were detected in response to Pool I than toPool II. Few to no spots were produced by splenocytes of gB-vaccinatedmice in response to stimulation with either of the peptide pools.

These data demonstrate that the HCMV DNA vaccine component pp65 wasexpressed in vivo at levels sufficient to induce cellular immuneresponses, either when it was administered alone or in combination, inthe VF-P1205-02A formulation.

Example 8 Confirmation of Immunogenicity Plasmids Encoding Human CMVAntigens General Experimental Procedure

The experimental procedure for the following example is as describedabove, with particular parameters and materials employed as describedherein.

Plasmids

As described above, constructs of the present invention, VR6365 and VR6368 were constructed by inserting the appropriate inserts into theexpression vector VR10551, and were formulated with poloxamerformulation VF-P1205-02A where noted.

Vaccination Regimen

Groups of nine, 6- to 8-week old female BALB/c mice(Harlan-Sprague-Dawley) received bilateral, intramuscular (rectusfemoris) injections (50 μl/leg) containing plasmid DNA encoding pp65,gB, or pp65 and gB with or without VF-P1205-02A on days 0, 21, and 49.Each mouse received 200 μg of DNA per vaccination. For formulationscontaining a single gB or pp65 coding plasmid, 100 μg of blank DNA(VCL10551), which served as a filler, was included. The effect of theblank DNA was tested by vaccinating mice with 100 μg of the singleplasmid DNAs delivered with or without VF-P1205-02A in the absence ofthe filler DNA. Serum samples were collected prior to the firstvaccination (day−1) and after each vaccination (days 20, 48, and 63) andgB-specific antibodies were measured by ELISA.

Recombinant gB Enzyme Linked Immunosorbent Assay (ELISA)

Sera were collected from the vaccinated mice, and anti-gB IgG titerswere determined using a recombinant CMV gB Enzyme Linked ImmunosorbentAssay (ELISA) as described in Example 7.

The anti-gB IgG titers in sera from mice vaccinated with VCL-6365,either alone or in combination with VCL-6368 are given below:

TABLE 13 Anti-gB IgG Titers Bleed 2 Bleed 3 (Day 48) (Day 63) Log₁₀ meantiter Log₁₀ mean titer Immunogen (range) (range) gB + pp65 in PBS 4.6 4.78 (4.4-4.7) (4.4-5.0) gB + pp65 + 4.7  4.96 VF-P1205-02A (4.1-5.0)(4.7-5.3) gB + neg. 4.98 5.25 control plasmid (3.8-5.3) (4.1-5.6) gB +neg. 4.87 5.14 control plasmid + (4.4-5.3) (4.7-5.6) VF-P1205-02A gB inPBS 4.82 5.15 (4.4-5.3) (4.7-5.6) gB + 4.73 5.1  VF-P1205-02A (4.4-5.0)(4.7-5.3)

Plasmid VCL6365 (gB) elicited the production of gB-specific antibodiesin mice that received three injections of the plasmids either alone orin combination. All mice vaccinated with VCL6365 had detectable anti-gBIgG titers after two injections. These data confirm the immunogenicityof the gB plasmid product in vivo when VCL6365 is delivered incombination with VCL6368 in the VF-P1205-02A formulation.

Example 9 Plasmid Encoding Human CMV IE1 is Immunogenic GeneralExperimental Procedure

The experimental procedure for the following example is as describedabove, with particular parameters and materials employed as describedherein.

Vaccination Regimen

Mice received bilateral, intramuscular injections into the rectusfemoris of the IE1 plasmid VR6250. The total DNA doses as shown belowwere each in a 100 μl volume in PBS, but was administered as two equalvolume injections, one into each rectus femoris muscle of each mouse.The negative control group contained 5 mice and all other groupscontained 10 mice. Mice received injections on days 0 and 14.Splenocytes were analyzed for IE1 reactivity by ELISpot assay in whichsplenocytes were stimulated with a pool of 98 overlapping 15mer peptides(overlapping by 11 amino acids) that span the entire IE1 protein encodedon the VR6250 construct. Splenocytes from the negative control groupwere harvested on day 24 and were analyzed for non-specific stimulationof IFN-γ secreting T-cells with the IE1 peptide pool. Splenocytes fromthe groups injected with IE1 DNA were harvested for analysis of antigenspecific, IFN-γ secreting, T-cell responses on days 27-29. Two spleensfrom each group were pooled for the assay. Two pools from each groupwere analyzed on days 27 and 28, one pool from each group was analyzedon day 29. The values reported below represent the average of 5splenocyte pools per experimental group.

IFN-γ ELISpot Assay

T cell responses to the DNA vaccines were determined by quantifying thenumber of splenocytes secreting IFN-γ in response to antigen-specificstimulation as measured by IFN-γ ELISpot assay. ImmunoSpot plates(Cellular Technology Limited, Cleveland, Ohio) were coated with ratanti-mouse IFN-γ monoclonal antibody (BD Biosciences, San Diego,Calif.), and blocked with RPMI-1640 medium. Splenocyte suspensions wereisolated from individual vaccinated mice and added to ELISpot plates at1×10⁶ or 3.3×10⁵cells/well in RPMI medium containing 5 μg/mL of each ofthe overlapping IE1 peptides as stimulating antigen. Control wellscontained 1×10⁶ splenocytes incubated in medium (no antigen). After a20-hour incubation at 37° C., captured IFN-γ was detected by thesequential addition of biotin-labeled rat anti-mouse IFN-γ monoclonalantibody and avidin-horseradish peroxidase. Spots produced by theconversion of the colorimetric substrate, 3-amino-9-ethylcarbazole(AEC), were quantified by an ImmunoSpot Analyzer (Cellular TechnologyLimited, Cleveland, Ohio). The results are expressed as spot formingunits (SFU) per 10⁶ cells.

TABLE 14 IE1 ELISpot Results DNA & 100 μg 1 μs 3 μg 10 μg 30 μg 100 μgDose Blank VR6250 VR6250 VR6250 VR6250 VR6250 SFU/10⁶ 6 5 77 289 367 501cells

The data shows that administering the IE1 plasmid VR6250 induced anantigen specific immune response, and that the immune response was DNAdose dependent. Additionally, this indirectly confirms that the IE1protein was expressed in vivo.

Example 10 Formulation Selection Studies

The potency of different vaccine formulations was evaluated in twoexperimental mouse immunogenicity studies using murine CMV M84. MurineCMV M84 is considered a homolog of the human CMV pp65, and thus servedas a surrogate for the pp65 antigen. The first study measured lipid doseresponses using a fixed quantity of DNA while the second study evaluatedclinically relevant doses of DNA by dose escalation.

Formulations

DMRIE/DOPE in a 1:1 molar ration was produced as a lipid film containing46.2% DMRIE and 53.8% DOPE by weight (5.14 mg total dried lipid). Priorto injection, the dried, mixed lipid film was hydrated in sterile waterfor injection to form cationic liposomes that were then added to DNA atthe appropriate concentration in 2×PBS. DNA was formulated withDMRIE/DOPE as follows:

TABLE 15 DNA Formulations DNA Concentration (mg/mL) DNA:Lipid* 0.5 2:11.0 4:1 3.0 10:1  *DNA (assigned MW = 333 gr/mol):cationic lipid molarratio

For the lipid dose response studies the DMRIE/DOPE formulations listedabove were diluted to a final vaccinating concentration of 0.5 mg/mL ofM84 DNA. For the DNA dose escalation studies the formulations were notdiluted prior to injection.

Poloxamer formulations for the lipid dose response study were producedwith 5 mg/mL of M84 DNA, 7.5 mg/mL of CRL 1005, and 0.3 mM ofbenzylalkonium chloride (BAK) surfactant. Prior to injection, theformulations for the lipid dose response study were diluted to a finalvaccinating concentration of 0.5 mg/mL of M84 DNA. In the DNA doseescalation studies, the formulations were produced with 3 mg/mL of theappropriate plasmid DNA, 4.5 mg/mL of CRL 1005, and 0.18 mM BAK. Theseformulations were not diluted prior to injection.

Vaccination Regimen

Groups of nine, six- to eight-week old BALB/c mice(Harlan-Sprague-Dawley) received bilateral (50 μL/leg) intramuscular(rectus femoris) injections of plasmid DNA formulated with DMRIE/DOPE orCRL 1005 in PBS. Control mice received DNA in PBS alone. All mice wereboosted on (approximately) days 21 and 49. Two weeks after the lastimmunization, splenocytes were harvested from three mice/group/day forthree sequential days, and antigen specific T cell responses weremeasured by IFN-γ ELISpot assay.

Cell Culture Media

Splenocyte cultures were grown in RPMI-1640 medium containing 25 mMHEPES buffer and L-glutamine and supplemented with 10% (v/v) FBS, 55 μMβ-mercaptoethanol, 100 U/mL of penicillin G sodium salt, and 100 μg/mLof streptomycin sulfate.

IFN-γ ELISpot Assay

T cell responses to the DNA vaccines were determined by quantifying thenumber of splenocytes secreting IFN-γ in response to antigen-specificstimulation as measured by IFN-γ ELISpot assay. ImmunoSpot plates(Cellular Technology Limited, Cleveland, Ohio) were coated with ratanti-mouse IFN-γ monoclonal antibody (BD Biosciences, San Diego,Calif.), and blocked with RPMI-1640 medium. Splenocyte suspensions wereproduced from individual vaccinated mice and seeded in ELISpot plates at1×10⁶, 3×10⁵, or 1×10⁵ cells/well in RPMI medium containing 1 μg/mL ofthe appropriate MHC class I-restricted peptide (M84, ²⁹⁷AYAGLFTPL³⁰⁵,(SEQ ID NO:32) Imgenex, San Diego, Calif.), 1 U/mL of recombinant murineIL-2 (Roche, Indianapolis, Ind.). Control wells contained 1×10⁶splenocytes incubated in medium with IL-2 only (no antigen). After a20-hour incubation at 37° C., captured IFN-γ was detected by thesequential addition of biotin-labeled rat anti-mouse IFN-γ monoclonalantibody and avidin-horseradish peroxidase. Spots produced by theconversion of the colorimetric substrate, 3-amino-9-ethylcarbazole(AEC), were quantified by an ImmunoSpot reader (Cellular TechnologyLimited, Cleveland, Ohio). Statistically significant differences betweenthe T cell responses of mice vaccinated with lipid- orpoloxamer-formulated DNA and naked DNA was determined using a Student'st-test with α=0.05.

The M84-specific CD8+ T cell responses of mice vaccinated with 50 μg ofM84 DNA formulated with DMRIE/DOPE (“DID”) at the DNA:lipid molar ratiosindicated, CRL 1005, or PBS alone are given below.

TABLE 16 CD8+ T Cell Responses Mean SFU/10⁶ Splenocytes VaccineFormulation CD8+ T cells PBS 299 2:1 D/D 243 4:1 D/D 179 10:1 D/D 299CRL 1005 344

The M84-specific CD8+ T cell responses of mice vaccinated withescalating doses of M84 DNA formulated with DMRIE/DOPE (D/D) at theDNA:lipid molar ratios indicated versus M84 DNA formulated with CRL 1005or PBS alone are given below.

TABLE 17 CD8+ T Cell Responses Vaccine Formulation Mean SFU/10⁶Splenocytes (DNA Dose) CD8+ T cells PBS (300 μg) 533 2:1 D/D (50 μg) 1844:1 D/D (100 μg) 158 10:1 D/D (300 μg) 243 CRL 1005 (300 μg) 416

Example 11 Experiments Employing HCMV Antigens Vaccination Regimen

Groups of nine, 6- to 8-week old female BALB/c mice(Harlan-Sprague-Dawley) received bilateral, intramuscular (rectusfemoris) injections (50 μl/leg) containing plasmid DNA encoding pp65,gB, or pp65 and gB with or without CRL 1005 (the VF-P1205-02Aformulation) on days 0 and 13. Each mouse received 200 μg of DNA pervaccination. For formulations containing a single gB or pp65 codingplasmid, 100 μg of blank DNA (VR10551) was added to yield 200 μg oftotal DNA. Beginning approximately three weeks after the primaryimmunization (on day 22), splenocytes were harvested from vaccinatedmice and pp65-specific T cell responses were measured by IFN-g ELISpotassay. Three ELISpot assays were performed: assay one measured theimmune response from a pool of splenocytes from three mice per group andassays two and three measured the immune response from a pool ofsplenocytes from two mice per group. The immune responses of theadditional two mice in each group were not measured in this series ofassays.

IFN-γ ELISpot Assay

T cell responses to DNA-encoded pp65 were determined by quantifying thenumber of splenocytes secreting IFN-γ in response to stimulation withpp65-derived peptides (Bio-Synthesis, Lewisville, Tex.). ImmunoSpotplates (Millipore, Billerica, Mass.) were coated with rat anti-mouseIFN-γ monoclonal antibody (BD Biosciences, San Diego, Calif.) andblocked with RPMI-1640 medium containing 25 mM HEPES buffer andL-glutamine and supplemented with 10% (v/v) heat inactivated FBS, 55 mMb-mercaptoethanol, 100 U/mL of penicillin G sodium salt, and 100 μg/mLof streptomycin sulfate (10% RPMI). Splenocyte suspensions were producedfrom vaccinated mice, resuspended in 10% RPMI medium at a density of2×10⁷ cells/mL, and seeded in triplicate wells of two separateImmunoSpot plates at a density of 5×10⁵ or 2.5×10⁵ cells/well.Splenocytes were stimulated with two separate pools of overlapping pp65peptides (one pool per plate) that, together, span the entire pp65protein and should include all possible T cell epitopes. Therefore, thetype of T cell (e.g., CD8+ or CD4+) that is producing IFN-γ in responseto the peptide stimulation cannot be distinguished by this assay method.Theoretically, these peptides can be presented in the context of class Ior class II MHC, thus stimulating both CD8+ and CD4+ T cells within thesame splenocyte preparation. The peptide pools contained 68 (pool I) or69 (pool II) peptides of 15 amino acids each (except one 13 amino acidpeptide in pool II), and each peptide was represented at a finalconcentration of 5 μg/mL in the assay well. Control wells contained5×10⁵ cells in medium only (no peptide antigen). After a 21-hourincubation at 37° C., captured IFN-γ was detected by the sequentialaddition of biotin-labeled rat anti-mouse IFN-γ monoclonal antibody (BDBiosciences, San Diego, Calif.) and avidin-horseradish peroxidase. Spotsproduced by the conversion of the colorimetric substrate,3-amino-9-ethylcarbazole (AEC), were quantified by an ImmunoSpot reader(Cellular Technology Limited, Cleveland, Ohio). Data are presented asthe number of Spot Forming Units (SFU), produced in response toantigen-specific stimulation, per million cells assayed. Theantigen-specific stimulation was calculated by subtracting the meannumber of spots in wells containing splenocytes incubated in mediumalone (the non-specific, background response) from the number of spotsin wells containing the identical splenocyte preparation incubated witha pool of pp65-derived peptides. Three replicate wells were used todetermine the mean non-specific background response. Each SFUcorresponds to one pp65-specific T cell. Due to the small sample size(n=3), a statistical analysis of the difference of the means was notperformed.

Experiment 1—See TABLES 18 and 19.

TABLE 18 T Cell Responses to CMV pp65 Peptide Pool I Mean Fold Increaseversus DNA Vaccine SFU/10⁶ Cells pp65 + gB pp65 + gB 170 — pp65 + gB +CRL 1005 705 4.1 pp65 + Blank 681 4.0 pp65 + Blank + CRL 1005 780 4.6gB + Blank 1 0 gB + Blank + CRL 1005 2 0

TABLE 19 T Cell Responses to CMV pp65 Peptide Pool II Mean Fold Increaseversus DNA Vaccine SFU/10⁶ Cells pp65 + gB pp65 + gB 80 — pp65 + gB +CRL 1005 208 2.6 pp65 + Blank 374 4.7 pp65 + Blank + CRL 1005 225 2.8gB + Blank 0 0 gB + Blank + CRL 1005 0 0

Experiment 2

The experiment above was repeated, and although the pp65+gB group hadresponses to peptide pool I that were 2.4-fold higher than that measuredin the study reported in detail above, the results were similar.

TABLE 20 T CELL RESPONSES TO CMV PP65 PEPTIDE POOL I Mean Fold Increaseversus DNA Vaccine SFU/10⁶ Cells pp65 + gB pp65 + gB 407 — pp65 + gB +CRL 1005 444 1.1 pp65 + Blank 435 1.1 pp65 + Blank + CRL 1005 762 1.9gB + Blank ND — gB + Blank + CRL 1005 ND —

TABLE 21 T Cell Responses to CMV pp65 Peptide Pool II Mean Fold Increaseversus DNA Vaccine SFU/10⁶ Cells pp65 + gB pp65 + gB 100 — pp65 + gB +CRL 1005 158 1.6 pp65 + Blank 140 1.4 pp65 + Blank + CRL 1005 225 2.3gB + Blank 0 — gB + Blank + CRL 1005 0 —

Experiment 3 Vaccination Regimen

Groups of nine, 6- to 8-week old female BALB/c mice(Harlan-Sprague-Dawley) received bilateral, intramuscular (rectusfemoris) injections (50 μl/leg) containing plasmid DNA encoding pp65,gB, or pp65 and gB with or without CRL 1005 (the VF-P1205-02Aformulation) on days 0, 21, and 49. Each mouse received 200 μg of DNAper vaccination. For formulations containing a single gB or pp65 codingplasmid, 100 μg of blank DNA (VCL10551) was added to yield a 200 μg doseof total DNA. The effect of the blank DNA was tested by vaccinating micewith 100 μg of the single plasmid DNAs delivered with or without CRL1005 in the absence of the blank DNA. Splenocytes were harvestedbeginning day 66 and pp65-specific T cell responses were analyzed byIFN-γ ELISpot as above. Based on previous results, no pp65-specific Tcell responses were anticipated for mice vaccinated with gB+blank DNA orgB+blank DNA+CRL 1005. Therefore, these mice were not evaluated in theELISpot assays. Statistically significant differences between the mean Tcell responses of vaccinated mice versus pp65+gB was determined using aStudent's t-test with α=0.05.

TABLE 22 T Cell Responses to CMV pp65 Peptide Pool I Mean Fold IncreaseDNA Vaccine SFU/10⁶ Cells versus pp65 + gB p-value pp65 + gB 783 — —pp65 + gB + CRL 1005 1360 1.7 0.03 pp65 + Blank 1265 1.6 0.02 pp65 +Blank + CRL 1005 1308 1.7 0.03 pp65 1184 1.5 NS pp65 + CRL 1005 1767 2.30.01 NS = not significant

TABLE 23 T Cell Responses to CMV pp65 Peptide Pool II Mean Fold IncreaseDNA Vaccine SFU/10⁶ Cells versus pp65 + gB p-value pp65 + gB 234 — —pp65 + gB + CRL 1005 544 2.3 0.04 pp65 + Blank 496 2.1 0.04 pp65 +Blank + CRL 1005 651 2.8 0.008 pp65 581 2.5 0.02 pp65 + CRL 1005 704 3.00.01

Example 12 Vaccine Combinations DNA and Protein Combination GeneralExperimental Procedure

The experimental procedure for the following example is as describedabove, with particular parameters and materials employed as describedherein.

Vaccination Regimen

BALB/c female mice, 6/group, were injected in each rectus femoris with20 μg of HCMV bivalent DNA vaccine in a 50 μl volume+/−poloxamerVF-P1205-02A (“02A”), DMRIE:DOPE, (“D/D”) and/or gB protein as indicatedbelow. Plasmid VR6365 encodes HCMV gB, plasmid VR6368 encodes HCMV pp65.Full-length gB protein purified from CHO cells was obtained from AustralBiologicals. (San Ramon, Calif.). Mice received injections on days 0 and14 and were bled for determination of gB antibody titers on day 13 andday 26. Splenocytes from two mice per group were harvested on days 26,27, and 28 for pp65 IFN-γ ELISpot analyses (splenocytes from individualmice were assayed, n=6 per group).

TABLE 24 Immunization Schedule Group DNA (total/injection/mouse) A 10 μgVR 6368 + 10 μg VR6365 + 02A B 10 μg VR 6368 + 10 μg VR6365 + 02A + 4.5μg gB protein C 10 μg VR 6368 + 10 μg VR6365 + 02A + 1.5 μg gB protein D10 μg VR 6368 + 10 μg VR6365 + 02A + 0.5 μg gB protein E 10 μg VR 6368 +10 μg VR6365 + D/D + 4.5 μg gB protein F 10 μg VR 6368 + 10 μg VR6365

Recombinant gB Enzyme Linked Immunosorbent Assay (ELISA)

The ELISA for detecting gB specific serum antibodies was performed with96 well Costar ½ well EIA plates coated with recombinant CMV gB at aconcentration of 0.1 μg/well in borate buffered saline (BBS) buffer.After coating with antigen, the plates were sealed and incubated at 4°C. overnight. Plates were washed 4× with BBS containing 0.1% Tween-20(BBST) using an automated plate washer. Non-specific binding was blockedby incubating plates for 1 hr at room temperature with 100 μL of assaybuffer (10% fetal calf serum in BBS). Blocking buffer was then decantedand serially diluted sera (diluted in assay buffer) added at 50 μl/well.Plates were sealed, incubated at room temperature for 2 hours, thenwashed 4× with BBS containing 0,1% Tween-20 (BBST) using an automatedplate washer. Goat anti-mouse IgG Fc specific secondary antibody dilutedat 1:5000 in assay buffer was added at 50 μl/well; plates were sealedand incubated at room temperature for 2 hours. Plates were washed 4×with BBS containing 0.1% Tween-20 (BBST) using an automated platewasher. Substrate, consisting of p-nitrophenylphosphate at 1 mg/ml in 50nM Sodium Bicarbonate buffer, pH 9.8 and MgCl₂ at 1 mM was added at 50μl/well, plates were sealed and incubated at room temperature for 60minutes. Absorbance of each well was determined at 405 nm. Endpointtiter=the reciprocal of the last dilution resulting in a mean absorbancevalue that is greater than or equal to twice the mean absorbance valueof background wells.

TABLE 25 Anti-gB IgG Titers and ELISpot Results HCMV gB HCMV gB HCMVpp65 antibody titers antibody titers Group SFU/10⁶ splenocytes Day 13Day 26 A 368 325 5867 B 576 467 22400 C 451 717 25600 D 260 500 14400 E523 1800 187733 F 465 75 1867

Adding gB protein to the bivalent gB, pp65 DNA vaccine formulated inpoloxamer increased the anti-gB antibody response up to 14-fold vs. thebivalent vaccine alone (bivalent vaccine+02A+1.5 μg gB protein (Group C)vs. bivalent vaccine alone (Group F), p=0.005) and up to 4-fold vs.bivalent DNA in poloxamer (bivalent vaccine+02A+1.5 μg gB protein (GroupC) vs. bivalent vaccine+02A (Group A), p=0.01). Adding gB protein to thebivalent DNA vaccine formulated in cationic lipid increased the anti-gBantibody response 101-fold vs. bivalent vaccine alone (bivalentvaccine+D/D+4.5 μg gB protein (Group E) vs. bivalent vaccine alone(Group F), p=0.00006) and 32-fold vs. bivalent DNA in poloxamer(bivalent vaccine+D/D+4.5 μg gB protein (Group E) vs. bivalentvaccine+02A (Group A), p=0.00005). The pp65 response was similar for allgroups indicating that combining protein with the bivalent DNA vaccineto improve the antibody component of the response did not decrease thecellular component of the response.

Example 13 Vaccine Combinations Trivalent Vaccine CombinationVaccination Regimen

Groups of 10 mice were injected in each rectus femoris with 50 μL of PBScontaining multiple DNA plasmids as shown below. Plasmid VR6365 encodesHCMV gB, Plasmid VR6368 encodes HCMV pp65, Plasmid VR6250 encodes HCMVIE1, and “blank” refers to an equivalent plasmid backbone but lackingany antigen coding sequence. All DNA was formulated with the “02A”poloxamer based formulation as described in Example 4. Two sets ofinjections were given on days 0 and 14. Serum was drawn at day 26 fordetermination of gB antibody titers.

TABLE 26 Immunization Schedule Group Dose (per leg) A 6.6 μgr VR6368(pp65) + 6.6 μgr VR6250 (IE1) + 6.6 μgr VR6365 (gB) B 6.6 μgr VR6368(pp65) + 6.6 μgr blank + 6.6 μgr VR6365 (gB) C 6.6 μgr blank + 6.6 μgrVR6250 (IE1) + 6.6 μgr VR6365 (gB)

Recombinant gB Enzyme Linked Immunosorbent Assay (ELISA)

Sera were collected from the vaccinated mice according to the regimendescribed in Example 7 above. Anti-gB IgG titers were determined using arecombinant CMV gB Enzyme Linked Immunosorbent Assay (ELISA), asdescribed in Example 12 above.

IFN-γ ELISpot Assay

Spleens were harvested for analysis of antigen specific, IFN-γsecreting, T-cell responses on days 27-29. Two spleens from each groupwere pooled for the assay. Two pools from each group were analyzed ondays 27 and 28, one pool from each group was analyzed on day 29.Splenocytes were processed and analyzed for pp65 reactivity by ELISpotassay as described in Example 7. Splenocytes were analyzed for IE1reactivity by ELISpot assay as described for pp65 ELISpot assay except,splenocytes were stimulated with a pool of 98 overlapping 15 merpeptides (overlapping by 11 amino acids) that span the entire IE1protein encoded on the VR6250 construct. (See Example 3).

TABLE 27 Anti-gB IgG Titers and ELISpot Results Analysis Group A Group BGroup C gB antibody titer 18,560 24,320 62,720 pp65 ELISpot 348 231 1(SFU/10⁶ splenocytes) IE1 ELISpot 218 1 319 (SFU/10⁶ splenocytes)

Earlier experiments showed that administering each antigen encoding DNAalone elicits an immune response in vivo. The present data show thateach antigen encoding DNA induces a specific immunological response whencombined with other antigens. Thus, combining the antigens andsimultaneously administering multiple antigen encoding DNAs allowsgeneration of immune responses to all the antigens simultaneously.

Example 14 Electrically-Assisted Plasmid Delivery

In vivo gene delivery may be enhanced through the application of briefelectrical pulses to injected tissues, a procedure referred to herein aselectrically-assisted plasmid delivery. See, e.g., Aihara, H. &Miyazaki, J. Nat. Biotechnol. 16:867-70 (1998); Mir, L. M. et al., Proc.Natl Acad. Sci. USA 96:4262-67 (1999); Hartikka, J. et al.; Mol. Ther.4:407-15 (2001); and Mir, L. M. et al.; Rizzuto, G. et al., Hum GeneTher 11:1891-900 (2000); Widera, G. et al, J. of Immuno. 164: 4635-4640(2000). The use of electrical pulses for cell electropermeabilizationhas been used to introduce foreign DNA into prokaryotic and eukaryoticcells in vitro. Cell permeabilization can also be achieved locally, invivo, using electrodes and optimal electrical parameters that arecompatible with cell survival.

The electroporation procedure can be performed with variouselectroporation devices. These devices include external plate typeelectrodes or invasive needle/rod electrodes and can possess twoelectrodes or multiple electrodes placed in an array. Distances betweenthe plate or needle electrodes can vary depending upon the number ofelectrodes, size of target area and treatment subject.

The TriGrid needle array, as described herein, is a three electrodearray comprising three elongate electrodes in the approximate shape of ageometric triangle. Needle arrays may include single, double, three,four, five, six or more needles arranged in various array formations.The electrodes are connected through conductive cables to a high voltageswitching device that is connected to a power supply.

The electrode array is placed into the muscle tissue, around the site ofnucleic acid injection, to a depth of approximately 3 mm to 3 cm. Thedepth of insertion varies depending upon the target tissue and size ofpatient receiving electroporation. After injection of foreign nucleicacid, such as plasmid DNA, and a period of time sufficient fordistribution of the nucleic acid, square wave electrical pulses areapplied to the tissue. The amplitude of each pulse ranges from about 100volts to about 1500 volts, e.g., about 100 volts, about 200 volts, about300 volts, about 400 volts, about 500 volts, about 600 volts, about 700volts, about 800 volts, about 900 volts, about 1000 volts, about 1100volts, about 1200 volts, about 1300 volts, about 1400 volts, or about1500 volts or about 1-1.5 kV/cm, based on the spacing betweenelectrodes. Each pulse has a duration of about 1 μs to about 1000 μs,e.g., about 1 μs, about 10 μs, about 50 μs, about 100 μs, about 200 μs,about 300 μs, about 400 μs, about 500 μs, about 600 μs, about 700 μs,about 800 μs, about 900 μs, or about 1000 μs, and a pulse frequency onthe order of about 1-10 Hz. The polarity of the pulses may be reversedduring the electroporation procedure by switching the connectors to thepulse generator. Pulses are repeated multiple times. The electroporationparameters (e.g. voltage amplitude, duration of pulse, number of pulses,depth of electrode insertion and frequency) will vary based on targettissue type, number of electrodes used and distance of electrodespacing, as would be understood by one of ordinary skill in the art.

Immediately after completion of the pulse regimen, subjects receivingelectroporation can be optionally treated with membrane stabilizingagents to prolong cell membrane permeability as a result of theelectroporation. Examples of membrane stabilizing agents include, butare not limited to, steroids (e.g. dexamethasone, methylprednisone andprogesterone), angiotensin II and vitamin E. A single dose ofdexamethasone, approximately 0.1 mg per kilogram of body weight, shouldbe sufficient to achieve a beneficial affect.

EAPD techniques such as electroporation can also be used for plasmidscontained in liposome formulations. The liposome plasmid suspension isadministered to the animal or patient and the site of injection istreated with a safe but effective electrical field generated, forexample, by a TriGrid needle array, or a four needle array. Theelectroporation may aid in plasmid delivery to the cell by destabilizingthe liposome bilayer so that membrane fusion between the liposome andthe target cellular structure occurs. Electroporation may also aid inplasmid delivery to the cell by triggering the release of the plasmid,in high concentrations, from the liposome at the surface of the targetcell so that the plasmid is driven across the cell membrane by aconcentration gradient via the pores created in the cell membrane as aresult of the electroporation.

Electroporation Study in Rabbits

Electroporation assisted DNA vaccine delivery was compared to DNAformulated with DMRIE:DOPE or CRL 1005 and DNA in PBS in a New ZealandWhite Rabbit model using CMV gB DNA. Rabbits (5 per group) were injectedin the tibialis muscle at 0 and 28 days with 50 μg DNA/500 μl/leg.Electroporation was performed immediately after injection using theBTX-ECM830 pulse generator with a 5 mm×8.6 mm 4 needle array at 200V(232 V/cm), 60 msec, 2 pulses, and 2 Hz.

Serum endpoint titers were measured at days 2, 14, 28, 42 and 56 by gBELISA. The ELISA for detecting gB specific serum antibodies wasperformed with 96 well Costar ½ well EIA plates coated with recombinantCMV gB at a concentration of 0.1 μg/well in borate buffered saline (BBS)buffer. After coating with antigen, the plates were sealed and incubatedat 4° C. overnight. Plates were washed 4× with BBS containing 0.1%Tween-20 (BBST) using an automated plate washer. Non-specific bindingwas blocked by incubating plates for 1 hr at room temperature with 100μL of assay buffer (10% fetal calf serum in BBS). Blocking buffer wasthen decanted and serially diluted sera (diluted in assay buffer) addedat 50 μl/well. Plates were sealed, incubated at room temperature for 2hours, then washed 4× with BBS containing 0.1% Tween-20 (BBST) using anautomated plate washer. Goat anti-rabbit IgG Fc specific secondaryantibody diluted at 1:5000 in assay buffer was added at 50 μl/well;plates were sealed and incubated at room temperature for 2 hours. Plateswere washed 4× with BBS containing 0.1% Tween-20 (BBST) using anautomated plate washer. Substrate, consisting of p-nitrophenylphosphateat 1 mg/ml in 50 nM Sodium Bicarbonate buffer, pH 9.8 and MgCl₂ at 1 mMwas added at 50 μl/well, plates were sealed and incubated at roomtemperature for 60 minutes. Absorbance was determined at 405 nm using anautomated 96 well plate reader. Endpoint titer=the reciprocal of thelast dilution resulting in a mean absorbance value that is greater thanor equal to twice the mean absorbance value of background wells.

TABLE 28 Serum endpoint titers Group Pre-bleed Day 14 Day 28 Day 42 Day56 CRL 1005 140 420 4830 46720 55040 DMRIE: 240 1360 5120 354987 218453DOPE PBS + 180 79360 221867 2703360 1884160 Electro- poration PBS 115135 2240 35840 35840

The mean anti-gB titers for the CRL 1005 group were slightly higher (upto 3 fold higher) than the titers for the PBS group, but the differenceswere not statistically significant at any time point. The mean anti-gBtiters for the DMRIE:DOPE group were 2-10 fold higher (p<0.05 at allpost-injection time points) than for gB DNA in PBS. Electroporationafter injection of gB DNA in PBS increased anti-gB titers 53-588 foldover gB DNA in PBS without electroporation (p<0.05 at all post-injectiontime points), 34-189 fold over the CRL 1005 group (p<0.05 at allpost-injection time points) and 8-58 fold over the DMRIE:DOPE group(p<0.05 at all post-injection time points).

Example 15 Treating Patients Using Compositions Comprising HumanCodon-Optimized HCMV pp65 and gB, and Fragments and Variants Thereof

The plasmid immunotherapeutic products are produced according to currentFDA Good Manufacturing Procedures (GMP) and are administered to humansubjects under an approved Investigational New Drug application.

Initial Studies

Thirty-two healthy adults are immunized by i.m. injection with 0.5 mg or2.5 mg each of plasmid DNA encoding optimized gB and pp65 on separateplasmids at 0, 2, and 8 weeks. Blood samples are drawn preimmunizationand at 2, 4, 8, 10, and 16 weeks for immunogenicity studies, includingELISpot assays to measure CD4+ and CD8+ T cell responses and antibodytiters for HCMV gB.

B. Administration to Hematopoetic Stem Cell (HSC) Transplant Donors andRecipients

Following the procedures above, healthy HSC donors are immunized withthe plasmid compositions at 4 and 2 weeks prior to donation.Immunogenicity assays are performed using blood drawn from the donors atpreimmunization, and every two weeks for 16 weeks post immunization.Recipients are divided into two groups. The first group receives the HSCfrom the immunized donors, but not be immunized themselves. The secondgroup receives the HSC from the immunized donors and are immunized withthe same plasmid compositions as the donors approximately four weeksafter HSC transplantation, and immunogenicity assays are performed atpretransplantation and every two weeks as above. Immunizations may berepeated every two weeks for both donors and recipients.

What is claimed is:
 1. An isolated polynucleotide comprising SEQ IDNO:5, or an isolated polynucleotide comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:6.
 2. The polynucleotideof claim 1, wherein the nucleotide sequence encoding SEQ ID NO:6 ishuman codon-optimized.
 3. The isolated polynucleotide of claim 1,further comprising a nucleotide sequence comprising SEQ ID NO:13.
 4. Thepolynucleotide of claim 1, further comprising an additional nucleotidesequence of a human codon-optimized gene encoding a humancytomegalovirus (CMV) polypeptide, immunogenic fragment, variant, orderivative thereof.
 5. The polynucleotide of claim 4, wherein the humanCMV polypeptide is IE1, an immunogenic fragment, variant, or derivativethereof.
 6. The polynucleotide of claim 4, wherein the additionalnucleotide sequence comprises SEQ ID NO:28 or
 30. 7. The polynucleotideof claim 1, wherein the polynucleotide is inserted into a vector orplasmid.
 8. A composition comprising: (a) an isolated polynucleotidecomprising SEQ ID NO:13 and (b) either an isolated polynucleotidecomprising SEQ ID NO:5 or an isolated polynucleotide comprising apolynucleotide encoding the amino acid sequence of SEQ ID NO:6.
 9. Thecomposition of claim 8, wherein the nucleotide sequence encoding SEQ IDNO:6 is human codon-optimized.
 10. The composition of claim 8, furthercomprising an isolated polynucleotide comprising a polynucleotideencoding IE1, an immunogenic fragment, variant, or derivative thereof.11. The composition of claim 10, wherein the polynucleotide comprisesSEQ ID NO:28 or
 30. 12. An isolated polynucleotide comprising SEQ IDNO:28 or
 30. 13. A composition comprising the plasmid VR6250, and anisolated polynucleotide comprising SEQ ID NO:5 or an isolatedpolynucleotide comprising a nucleotide sequence encoding the amino acidsequence of SEQ ID NO:6.
 14. A composition comprising a polypeptide withthe amino acid sequence of SEQ ID NO:6 and an IE1 polypeptide,immunogenic fragment, variant, or derivative thereof.
 15. A method ofeliciting an immune response to human CMV in a human or in a humantissue comprising administering the polynucleotide of claim
 1. 16. Themethod of claim 15, wherein the immune response is a cellular immuneresponse and/or a humoral immune response.
 17. The method of claim 15,wherein the polynucleotide is formulated with(+/−)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE).
 18. The method of claim 15, further comprising anucleotide sequence comprising SEQ ID NO:13.
 19. The method of claim 15,further comprising a nucleotide sequence encoding an IE1 polypeptide,immunogenic fragment, variant, or derivative thereof.
 20. The method ofclaim 19, wherein the nucleotide sequence is SEQ ID NO:28 or 30.